Historical perspectives of white rust caused by Albugo candida in oilseed Brassica

M. Borhan

Albugo candida (Pers. Ex. Lev.) Kuntze is a wide spread pathogen of cruciferous crops causing heavy yield losses all over the world. Molecular and phylogenetic studies of the family Albuginaceae revealed four distinct lineages: Albugo s.str., Albugo s.l., Pustula s.l. and Wilsoniana s.l. It’s host range is more than 300 hosts. The host specificity of A. candida has been recorded from more than eight countries of the world. Studies on host-pathogen interaction, fine structures of hyphae, mycelium, haustoria, sporangia, zoospores and oospores have been conducted through histopathology, electron microscopy, scanning electron microscopy and transmission electron microcopy. The pathogen survives through mycelium, sporangia and oospores. Germination of sporangia and oospores has been determined. Biochemical host-pathogen interaction studies have been conducted. Studies on identification and cloning of plant defense resistance genes are in progress. Genome sequencing of A. candida and A. l...

Journal of Oilseed Brassica, 5 (Special) : 1-41, Jan 2014 Journal of Oilseed Brassica, 5 (special) : Jan 2014 1 Historical perspectives of white rust caused by Albugo candida in Oilseed Brassica P.D. Meena1, P.R. Verma2, G.S. Saharan3, and M. Hossein Borhan4 1 Directorate of Rapeseed-Mustard Research, Bharatpur-321 303, Rajasthan, India Email: pdmeena@gmail.com 2 Retired Senior Oilseed Pathologist, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK., S7N OX2 Canada, Email: prithwirajverma@gmail.com 3 Former Professor & Head, Department of Plant Pathology, CCSHAU, Hisar-125004, Haryana, India, Email: gssaharan675@gmail.com 4 Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK., S7N 0X2, Canada, Email:Hossein.Borhan@agr.gc.ca Abstract Albugo candida (Pers. Ex. Lev.) Kuntze is a wide spread pathogen of cruciferous crops causing heavy yield losses all over the world. Molecular and phylogenetic studies of the family Albuginaceae revealed four distinct lineages: Albugo s.str., Albugo s.l., Pustula s.l. and Wilsoniana s.l. It’s host range is more than 300 hosts. The host specificity of A. candida has been recorded from more than eight countries of the world. Studies on host-pathogen interaction, fine structures of hyphae, mycelium, haustoria, sporangia, zoospores and oospores have been conducted through histopathology, electron microscopy, scanning electron microscopy and transmission electron microcopy. The pathogen survives through mycelium, sporangia and oospores. Germination of sporangia and oospores has been determined. Biochemical host-pathogen interaction studies have been conducted. Studies on identification and cloning of plant defense resistance genes are in progress. Genome sequencing of A. candida and A. laibachii have been made. Very useful and reproducible techniques have been developed on the aspects viz., growth chamber inoculation, oospore germination, induction of stag-heads, detached leaf culture, In vitro callus culture, temperature effects on disease development and oospore formation, process of infection, association of white rust and downy mildew, pathogenic variability, virulence spectrum, host resistance, genetic of host-parasite interaction, slow white rusting and chemical control. Future research areas have been suggested. Keywords : White Rust, Albugo candida, Oil Seed Brassica 1. Introduction Albugo candida (Pers. Ex. Lev.) Kuntze. (A. cruciferarum S.F. Gray), a member of the family Albuginaceae in the order Albugonales of class Peronosporomycetes is an obligate parasite responsible for causing white rust (WR) disease of many cruciferous crops (Saharan and Verma, 1992). Local infection produces white to cream coloured pustules on leaves, stems and pods, while general or flower bud infection (Verma and Petrie, 1980) causes extensive distortion, hypertrophy, hyperplasia and sterility of inflorescences generally called “stagheads”. The staghead phase (SP) accounts for most of the yield loss attributed to this disease. Depending on the severity of both foliar and SP of the disease, the percent yield losses ranging from 1-60 % in Polish or Turnip rape (Brassica rapa L.) in Canada (Berkenkamp, 1972; Petrie and Venterpool, 1974; Harper and Pittman, 1974; Petrie, 1973), from 23-89.8 % in Indian mustard [B. juncea (L.) Czern and Coss] in India (Bains and Jhooty, 1979; Lakra and Saharan, 1989a), and from 5-10 % in Australia (Barbetti, 1981; Barbetti and Carter, 2 Journal of Oilseed Brassica, 5 (special) : Jan 2014 1986) have been reported; substantial yield losses in radish (Raphanus sativus L.) have also been reported (Kadow and Anderson, 1940; Williams and Pound, 1963). Although Canadian and European B. napus cultivars are not attacked in some countries, many cultivars of this species grown in China are susceptible (Fan et. al., 1983). The wide range of yield losses caused by this disease in many host species needs assessment of genotypes under suitable environmental conditions (Saharan, 2010). In the present manuscript the progress made in white rust research on biology, ecology, epidemiology and management of A. candida on oilseed Brassica and future priority research areas have been discussed. 2. Taxonomy and nomenclature The first species of Albugo described by Gmelin in 1792 as Aecidium candidum was later placed in genus Uredo, subgenus Albugo by Persoon in 1801. Based on differences in symptom development, Persoon (1801) described two different species of white blister rust, with Uredo candida, and subdivided into three varieties, parasitic to Brassicaceae and Asteraceae. After a few years, Albugo was established as an independent genus by de Roussel (1806), although Gray (1821) is often still given as the author for this genus. De Candolle (1806) added the species Uredo portulaceae (now Wilsoniana portulacae), and Uredo candida beta tragopogi to species rank (Uredo tragopogi, now Pustula tragopogonis) and renamed Uredo candida cruciferarum. Leveille (1847) described the genus Cystopus, and later de Bary (1863) described the sexual state of Albugo, adopting the generic name Cystopus. Albugo has been typified by Kuntze (1891), who gave Uredo candida (Pers) Pers. as the type species. Before Biga (1955) pointed out that names of sexual form have no antecedence over anamorphs in the class Oomycetes, many researchers considered white blister rusts to be members of the superfluous genus Cystopus (Wakefield, 1927), although the older genus name, ‘Albugo’ also persisted. Subsequently, in the early 20th century, numerous other species of genus Albugo were described. Wilson (1907) and Biga (1955), respectively recorded 13 and 30 species of this genus about 50 years later (Mukerji, 1975). The recent key to the genus Albugo published by Choi and Priest in 1995 recognised only 10 species of genus Albugo. Until molecular phylogenetic studies of the Albuginaceae became possible, Albugo was generally treated as a member of the Peronosporales (Dick, 2001), in which it was placed along with the second group of obligate plant parasites, the downy mildews (DM). The Albuginaceae family contains four distinct lineages: Albugo s.str., parasitic to Brassicales; Albugo s.l., parasitic to Convolvulaceae; Pustula s.l., parasitic to Asterales, and Wilsoniana s.l., parasitic to Caryophyllales. Albugo cruciferarum is regarded as a synonym of A. candida (Choi et al., 2007). Till now, the white blister pathogen on oilseed rape has been considered A. candida (Farr and Rossman, 2010). The high degree of genetic diversity exhibited within A. candida complex warrants their division into several distinct species (Choi et al., 2006). 3. Host range The first record of A. candida on Brassicaceae seems to be by Colmeiro (1867). Albugo candida has been reported in Brassicaceous hosts over widely different geographical areas of the world with host range of some 63 genera and 241 species (Biga, 1955; Saharan and Verma, 1992; Choi et al., 2007; Farr et al., 1989). According to the USDAARS Systemic Botany and Mycology Laboratory, A. candida was recorded on more than 300 hosts (Farr et al., 2004). Based on recent molecular phylogenetic investigations, A. candida has an extraordinarily broad host range, extending from numerous genera of the families Brassicaceae to Cleomaceae, and Fabales to Capparoceae (Choi et al., 2006, 2007, 2008, 2009, 2011a). A. candida and A. tragopogonis each may consist of several distinct lineages (Voglmayr and Riethmuller, 2006). The host specificity of A. candida has been recorded from Australia (Kaur et al., 2008), Britain (Happer, 1933), Canada (Verma et al., 1975), Germany Journal of Oilseed Brassica, 5 (special) : Jan 2014 (Eberhardt, 1904), India (Saharan, 2010), Japan (Hiura, 1930), Romania (Savulescu and Rayes, 1930), and U.S.A. (Pound and Williams, 1963). Albugo candida isolates from Brassica can infect Amaranthus viridis (Amaranthaceae), Cleomev viscosa (Capparaceae, now included in Brassicaceae; APG, 2003), as well as B. rapa var. Rapa (Khunti et al., 2000). Recently, Saharan (2010) has listed all pathotypes reported globally (Table 1). 4. Geographical distribution White rust on cultivated oilseed Brassicas and other hosts have been reported worldwide. Countries where the disease occurs include the U.K. (Berkeley, 1848), U.S.A. (Walker, 1957), Brazil (Viegas and Teixeira, 1943), Canada (Greelman, 1963; Petrie, 1973), Germany (Klemm, 1938); India (Chowdhary, 1944), Japan (Hirata, 1954), Pakistan (Perwaiz et al., 1969), Palestine (Rayss, 1938), Romania (Savulescu, 1946), Turkey (Bremer et al., 1947), Fiji (Parham, 1942), New Zealand (Hammett, 1969), China (Zhang et al., 1984) and Korea (Choi et al., 2011a). White rust on sunflower occurs in Russia (Novotel’Nova, 1962), Uruguay (Sackston, 1957), Argentina (Sarasola, 1942), Australia (Middleton, 1971; Stovold, and Moore, 1972), and in many other countries (Kajomchaiyakul and Brown, 1976). White rust of salsify occurs in Australia, Canada, U.S.A., S. America, Europe, Asia and Africa (Wilson, 1907), and on water spinach occurs in India, Hong Kong (Ho and Edie. 1969; Safeefulla, and Thirumalachar, 1953), and also in Texas (Wiant, 1937; Williams and Pound, 1963). 5. Structures and reproduction Studies on host-pathogen-interaction, fine structures of hyphae, mycelium, sporangia, zoospores and oospores have been conducted through histopathology using electron microscopy, scanning electron microscopy and transmission electron microscopy (Berlin and Bown, 1964; Davison, 1968; Coffey, 1975; Hughes, 1971; Khan, 1976, 1977; Tewari et al., 1980; Kaur et al., 1984; Baka, 2008). The members of the Albuginaceae are distinguished from those of related families by the formation of the asexual sporangia in basipetal chains. 3 5.1 Mycelium The non-septate and intercellular mycelium of Albugo species feeds by means of globose or knob-shaped intracellular haustoria, one to several in each host cells (Verma et al., 1975). The detail of haustorial formation and development has been given by Berlin and Bowen (1964); Coffey (1975); Davison (1968); Fraymouth (1956), and Wager (1896). 5.2 Asexual organs 5.2.1 Sporangiophore The sporangiophores are short, hyaline, clavate, thickwalled, especially towards the base, 30-45 x 15-18 µm diameter, basally branched, club-shaped and give rise to simple chains of sporangia. The number of sporangia produced is indefinite. They are formed in basipetal succession; that is, the sporangiophore forms a cross-wall or septum, cutting off that portion which is to become a sporangium. The sporangiophore increases in length, a second sporangium is cut off, and the process continues, resulting in the simple chains of multinucleate sporangia. 5.2.2 Sporangia The number of sporangia produced is indefinite in basipetal succession; that is, the sporangiophore forms a cross-wall or septum, cutting off that portion which is to become a sporangium, which is globose to oval, hyaline with uniform thin wall, and 12-18 µ m diameter. As sporangial production continues, the older, terminal portions of the chain breaks, releasing the individual sporangia. The sporangia germinate by the formation of zoospores and, on rare occasions, by means of a germ tube (Heald, 1926; Wager, 1896; Walker, 1957; Zalewski, 1883). 5.2.3 Zoospores Sporangia absorb water and swell, develop vacuoles in the granular protoplasm, and finally 4-12 uninuleate polyhedral portions of the protoplasm are delineated by fine lines. In the mean time, an obtuse papilla is formed at one side of the sporangium, which produces zoospores. The zoospores, still immobile, emerge usually one by one, with final cleavage following complete emergence 4 Journal of Oilseed Brassica, 5 (special) : Jan 2014 of the sporangium’s contents. The flagella soon become apparent by an oscillatory motion of the entire zoospore mass. These single-nucleated spores formed in sporangia are released only in aqueous environment. The slightly concave-convex zoospore contains a disc-like vacuole on one side, near which are attached two flagella, one short and one long, by which the zoospore soon detaches itself from the mass and swims away if liquid is present. They have one tinsel flagellum, and one whiplash flagellum. Only the tinsel flagellum has distinctive flagellar hairs. Zoospore formation occurs within minutes and is considered one of the fastest developmental processes in any biological system. Once released from the sporangium, zoospores exhibit chemotactic, electrotaxis, and autotaxis or auto-aggregation responses to target new hosts for infection (Walker and West, 2007). Zoospores soon come to rest, retract their flagella, encyst and germinate by the formation of a germ tube. If germination occurs on a susceptible host, the germ tube penetrates through stomata to form an intercellular mycelium (Heald, 1926; Wager, 1896; Walker, 1957). the periplasm and comes in contact with the egg cell or ooplasm. The antheridial or male nuclei are discharged through this tube into the egg cell. In the uninucleate egg, the female nucleus fuses with a single male nucleus, where as in the multinucleate egg, female and male nuclei fuse in pairs. This nuclear union constitutes the process of fertilization (Heald, 1926; Walker, 1957). Following fertilization, the egg is gradually transformed into a thick-walled oospore. The periplasm is absorbed, the oospore wall darkens and thickens, and develops a characteristic external ridges, reticulations or knobs, while the interior of the oospore becomes filled with an abundance of reserve food in the form of oily or fatty globules. The fully developed oospore lies within the old empty oogonial cell. The oospores are released only by weathering and decay of the host tissues (Heald, 1926). The characteristics of oospores are useful criteria for distinguishing species of Albugo, in which the epispore is tuberculate or ridged, and is a more specialized group, where there is complete development of the epispore with cytological phenomena (Zalewski 1883; Stevens, 1901). 5.3 Sexual organs 6. Survival 6.1 Mycelium The oogonia and antheridia are formed from the mycelium in the intercellular spaces of the host, particularly in a systemically invaded tissue (Wager, 1896). Oogonia are globose, terminal or intercalary, each contains upto 100 nuclei and its contents clearly defined into a peripheral zone of periplasm and a single central oosphere. Antheridia are clavate, each contains 6 to 12 nuclei, and are applied to the sides of an oogonium (Heald, 1926, Heim, 1959, Walker, 1957). 5.3.1 Gametogenesis, fertilization, and oospore formation One or more antheridia come to occupy a position close to an oogonium. There are two types of egg organization within an oogonium. In A. candida, the protoplast becomes differentiated into a peripheral or extemal zone, the periplasm, which contains many nuclei, and a central mass, the egg cell or ooplasm, which contains a single nucleus. The antheridium, which is a multinucleate cell, produces a short, tubelike outgrowth, the fertilization tube, which penetrates It is believed that in perennial hosts such as horseradish, the mycelium is capable of overwintering in the infected crowns and lateral roots (Endo and Linn, 1960; Kadow and Anderson, 1940; Walker, 1957). Remaining dormant during the winter, the mycelium resumes its activity and grows into the new shoots the host produces in the spring. 6.2 Sporangia At 30°C temperature, viability of sporangia is lost after 4 h when attached, and after 2 h when detached from host tissues (Lakra et al., 1989). They observed that sporangia of A. candida can survive for 4.5 days at 15°C on detached-infected B. juncea leaves, but loses their viability after 18 h if separated and incubated without host tissues. However, sporangia can be stored for 105 days at -40°C as a dry powdered mass. 6.3 Oospores Oospores are formed in the hypertrophied tissues Journal of Oilseed Brassica, 5 (special) : Jan 2014 (leaves, stems, inflorescences, pods, roots) of infected host plants. Overwintered oospores in infected plant debris in soil function as the source of primary inoculum of the pathogen (Butler, 1918; Butler and Jones, 1961; Chupp, 1925; Kadow and Anderson, 1940; Verma et al., 1975; Walker, 1957). Oospores have also been observed in naturally infected senesced leaves of B. juncea and B. rapa var. Toria. Lakra and Saharan (1989b) estimated 8.75 x 105 oospores in one gram of hypertrophied cup-shaped leaves, and 21.85 x 105 in one gram of hypertrophied staghead portions. Verma and Petrie (1975) found that oospores can remain viable for over 20 years under dry storage conditions. Petrie (1975) reported 1500 oospores per gram seed of rapeseed and reported the possibility of survival and spread of the pathogen by means of oospores carried extrnally on seeds. According to Tewari and Skoropad (1977), oospores have a highly differentiated, 5- layered cell wall and that their greater longevity is probably due to the heavily fortified cell wall. 7. Spore germination 7.1 Oospores de Bary (1866) first observed germination of Albugo oospores via asessile vesicle. Vanterpool (1959) confirmed this and described a second mode of germination by means of a terminal vesicle; however, maximum germination was only 4% and its occurrence was unpredictable. Petrie and Verma (1974) and Verma and Petrie (1975) described a very reliable and reproducible technique for germination of A. candida oospores. Oospores germinated by the production of one or two simple or branched germ tubes, by the release of zoospores from vesicles formed at the ends of germ tubes (terminal vesicles), and by the release of zoospores from sessile vesicles. Germination by sessile vesicles was the most common. Verma and Bhowmik (1988) observed that the treatment of oospores with 200 ppm KMn04 for 10 minutes induced increased germination. Oospores do not appear to require any dormancy period. Recently gut enzymes (1% b-glucuronidase and arylsulfatase, Sigma make) were used in studies for germination of the oospores from hypertrophied plant tissue (Meena and Sharma, 2012). 5 7.2 Sporangia Sporangial germination in A. candida was studied by several researchers. In 1911, Melhus reviewed the earlier work on sporangial germination. Prevost (1807), and De Bary (1860) found that sporangial germination occurs via the production of zoospores. Harter and Weimer (1929) stated that sporangia may germinate by the direct production of germ tubes, but germination via zoospores was more frequent. Eberhardt (1904), Melhus (1911), and Napper (1933) found that sporangia of A. candida germinate invariably by the production of zoospores; which was confirmed by Lakra et al. (1989). De Bary (1860), and Melhus (1911) reported that sporangia did not germinate above 25°C or below 0°C; the best germination was at lower temperatures. Napper (1933) did not observe sporangial germination above 20°C. Melhus (1911) suggested 10°C as the optimum temperature for sporangial germination, but Napper (1933) found that germination takes place as readily at 1-18°C. Endo and Linn (1960) reported the overall optimum temperature range for sporangial germination to be 15-20°C, with maximum germination occurring between 0 and 28°C. However, Lakra and Saharan (1988b), and Lakra et al. (1989) observed >75% sporangial germination in A. candida at 12-14°C after 8 h incubation. Sporangia ceased to produce zoospores below 6°C and above 22°C. Sporangial germination started after 4 h and reached their maximum 8 h after incubation. A quadratic equation, Y -103.16 + 26.99 x -1.01 x2, where Y = % sporangial germination and x = temperature in °C was proposed to estimate the frequency of sporangial germination of A. candida from B. juncea at any known temperature. The variation in the cardinal temperatures for sporangial germination among different studies is probably due to the involvement of different host specific biological races of A. candida. Germination of A. candida sporangia from naturally-infected B. juncea and B. rapa var. Toria leaves occurred within one hour at 13°C. Although Melhus (1911), and Holliday (1980) reported that sporangial germination is not affected by light or darkness, Lakra et al. (1989) demonstrated that exposure to light of 150 µEM-2s-1 slightly 6 Journal of Oilseed Brassica, 5 (special) : Jan 2014 delays sporangial germination in A. candida infecting B. juncea. Melhus (1911) found that sporangia germinated readily in both saturated and non-saturated atmosphere, while Lakra and Saharan (1988b), and Lakra et al. (1989) found that a film of free water is essential for germination of sporangia. Melhus (1911), and Napper (1933) found that chilling and a reduction of 30% water content in sporangia were essential for germination. Lakra et al. (1989), however, states that it is not a prerequisite, since up to 75% of sporangia germinated without chilling or dehydration. According to Uppal (1926), sporangia of A. candida require oxygen for germination. Takeshita (1954) reported that sporangia of A. candida from horseradish germinated best at pH 4.5-7.5 at 10-20°C. Light did not affect germination. However, Endo and Linn (1960) found that sporangia of A. candida from horseradish require pH of 3.5-9.5 with an optimum of about 6.5; optimum temperature range was 15-20°C. Only a few studies have been carried out on sporangial germination of species other than A. candida. Edie and Ho (1970) demonstrated that although the sporangial germination in A. ipomoeae-aquaticae is nearly identical with that of other Albugo species with regard to the method of sporangial germination and host penetration, it requires a slightly higher germination temperature in the range of 12-30°C with an optimum of about 25°C. However, Saffeefulla and Thirumalachar (1953) mentioned that sporangia germinated at 15°C, but not at 24°C. Sporangia of A. ipomoeae-panduratae germinate at 8-25°C (Harter, and Weimer, 1929) and optimum of 12-14°C. Sporangia of A. tragopogonis germinate at 4-35°C with an optimum range of 4-15°C. Encysted zoospores germinate best at 10°C (Kajomchaiyakul and Brown, 1976). Sporangial germination of A. tragopogonis from Senecio squandus occurs at 5-15°C, with an optimum of 10-15°C and very little germination occurs at 20°C (Whipps, and Cooke, 1978a, 1978b). Sporangia of A. bliti germinate at a temperature range of 2-25°C, but optimum at 18°C (Mishra and Chona, 1963). Chilling of sporangia, increases germination but mature sporangia from just-opened pustules, or those naturally-detached, germinated best. Sporangia of A. occidentalis germinate at 2-25°C with an optimum near 12°C (Raabe and Pound, 1952). Light, water content of sporangia, and pH also have little effect on sporangial germination. 8. Fine structures Electron microscopy, particularly when used in association with physiological, biochemical and genetic studies, provides valuable information on the complex relationships which exist between host and pathogen. The fine structures of A. candida were studied by Berlin and Bowen (1964a, b), Davison (1968) and Coffey (1975). 8.1 Haustoria : The small stalked capitate haustoria of Albugo are connected to the much larger haustorial mother cell by a slender cylindrical neck. Haustoria contain mitochondria with tubular cristae, ribosomes and occasional cisternae of rough endoplasmic reticulum. Nuclei and perinuclear dictyosomes, although present in the mother cells, are absent in the haustoria. The fungal plasma membrane and cell wall are continuous from an intercellular hypha to the haustorium except that there is no evidence of a fungal cell wall around a portion of the haustorial stalk proximal to the haustorial head (Saharan and Verma, 1992). In the host mesophyll cell, the haustorium is invariably surrounded by host plasma membrane and/or a thin layer of host cytoplasm. The host cell wall invaginates at the point of haustorial penetration to form a short sheath around the penetration site, but the host cell wall is absent from rest of the haustorium. A collar consisting of fibrillar material is commonly found around the proximal portion of the neck. An electron-opaque capsulation lies between the haustorium and the host plasma membrane, and extends into the penetration region between the sheath and the fungal cell wall. An electron-opaque sheath surrounds the thin wall of the haustorial body, but is absent from the neck region. A series of tubules is continuous with the invaginated host plasma membrane which surrounds the haustorial body. These tubules contain an electron-dense core similar in appearance to, and continuous with, the sheath matrix. Host dictyosomes and their secretory vesicles are not involved in formation of the haustorial sheath Journal of Oilseed Brassica, 5 (special) : Jan 2014 (Saharan and Verma, 1992). A constant feature of the haustorial apparatus is the association of flattened cistenae of host endoplasmic reticulum with the distal portion of the haustorial neck. Woods and Gay (1983) provide evidence for a neckband delimiting structural and physiological regions of the host plasma membrane associated with haustoria of A. candida. Coffey (1983) demonstrated cytochemical specialization at the haustorial interface of A. candida. Soylu (2004) observed that ultrastructural nature of the haustorium produced in Arabidopsis clearly differ from the DM, rust or the powdery mildew (PM) fungi. 8.2 Sporangia : In sporangia, the paramural bodies are formed by elaborations of the plasma membrane and break away from the plasma membrane and undergo autodigestion. In vegetative hyphae, the tubules and lamellae of paramural bodies break up into vesicles and are finally sequestered into the cell wall (Khan, 1976, 1977). The surface layer of the cell wall of the sporangia and sporangiophores of A. candida is composed of a series of lamellae. Evidence from freeze-fracture, freeze-etch, and single-stage replicas demonstrated that the lamellae are bilayered, an organization associated with the presence of lipids. This multilamellate layer on the surface of the cell wall facilitates air dispersal and protects the sporangia from desiccation (Tewari et al., 1980). In Albugo sporangia are produced in basipetal chains at the apices of sporangiophores and are released by the dissolution of the septa that delimit them. Hughes (1971) suggested that sporangiophores of Albugo produce sporangial chains by percurrent proliferation, and they are “apparently the morphological equivalents of annellophores (annellides)” (Hughes, 1971). A sporangial initial buds out from a fixed locus at the tip of the sporangiophore. After reaching a certain size, it is delimited by a basal septum and converted into a sporangium. A new initial grows out from the sporogenous locus, pushing the newly formed sporangium upward. By repetition of this process, a basipital chain of sporangia is formed. Both layers of the sporangiophore wall grow out and take part in forming the sporangial wall. In conidium ontogeny this mode of development is called holoblastic. 7 During sporangial formation in A. candida the sporangiophores do not increase in length; however, abnormally long sporangiophores are sometimes seen among the smaller, regular ones. There are no annellations on the sporangiophore surface and no increase in the thickness of the sporangiophore wall at its apex. Thus, none of the characteristics that have been shown to be associated with percurrent proliferation are present during the development of sporangia in Albugo (Khan, 1977). In maturing sporangia a burst of activity was observed by Khan (1976). Even after formation of sporangia, the numbers of mitochondria and the amounts of endoplasmic reticulum increase. Perinuclear vesicles and smooth surface cistenae differentiate into well developed Golgi apparatuses, which remain secretory until complete maturation of sporangia. Maturing sporangia have autophagic vacuoles containing various cell organelles. Nuclear degeneration and mitosis proceed simultaneously. All activities decline towards the end of sporangial maturation. 8.3 Oospores The structure and development of oospores of A. candida in the stagheads on rapeseed (B. rapa) were investigated by light microscopy, transmission electron microscopy of ultrathin sections and scanning electron microscopy (Tewari and Skoropad, 1977). A reaction zone forms on the oogonial wall at the point of contact by the fertilization tube of the antheridium. The oospore has a highly differentiated, five-layered cell wall. The periplasm appears to play an active role in the deposition of the oospore cell wall. The contents of the periplasm do not disappear after maturation of the oospore; instead, they form a persistent material between it and the oogonial wall. Hence, functionally, the oospore wall complex has two additional layers which may contribute to the longevity of the oospore. In a histochemical study of cytoplasmic changes during wall layer formation on the oospore of A. candida, Kaur et al. (1984) reported that the young multinucleate oogonium is double-walled. The oospore nuclei are large and prominent, and have an outer shell or sheath of proteinaceous material surrounding a central core of nucleoplasm. The first wall of the fertilized oospore is laid at the interphase 8 Journal of Oilseed Brassica, 5 (special) : Jan 2014 of the periplasm and the ooplasm. Subsequent wall layers are formed both on the inner and outer side of the first oospore wall. The second oospore wall is formed just internal to the first one. The third wall of the oospore is formed external to the first one and appears ridged. The last wall to be formed is the Innermost one which completely surrounds the central ooplasm. This wall layer is callosic in nature.Oospore morphology is basically reticulate. 9. Biochemistry of host pathogen interaction Biochemical studies of the growth and survival of a pathogen, and of the changes it induces in its host can ultimately lead to a better understanding of epidemiology, disease development and control. With a few exceptions, such studies on WR lag far behind those for diseases caused by other major groups of biotrophs. Ideal prerequisites for meaningful studies of the biochemistry of hostparasite interaction are a) a clear understanding of the genetic control of virulence and avirulence in the parasite and of susceptibility and resistance in the host, b) precise histological and cytological descriptions of spore germination, infection and the establishment and development of infection, and c) the availability of methods for growing the parasite alone and in combination with its host under controlled conditions. Unfortunately, these criteria have not been fully satisfied for any WR disease. Reduction in sugar content was proportionate to the disease severity, and maximum reduction was observed in the infected leaves. Total free amino acids increased after infection in all the infected plant parts, and this increase was proportionate to the disease severity (Singh, 2005). 9.1 Carbohydrate metabolism and respiration A number of reports indicate that the respiration rates of tissues infected by members of the Albuginaceae also rise dramatically (Black et al., 1968, Williams and Pound, 1964). Long and Cooke (1974) suggested that host-fungus movement of carbohydrates in Albugo-Senecio squalidus system is maintained by hydrolysis of host sucrose and uptake of hexoses, followed by accumulation of trehalose within the mycelium and spores. Trehalose was synthesized within pustules by the fungus but no acyclic polyols were found. Accumulation of hexoses around pustules together with increased hydrolysis of exogenous sucrose by pustular material indicated increased invertase activity within infected tissues. Accumulation of darkfixed carbon compounds in WR pustules of Senecio squalidus infected with A. tragopogonis has been reported (Thomton and Cooke. 1970). Quantitative imaging of chlorophyll fluorescence revealed that the rate of photosynthesis declined progressively in the invaded regions of the leaf. Images of nonphotochemical fluorescence quenching (NPQ) suggested that the capacity of the Calvin cycle had been reduced in infected regions, and that there was a complex metabolic heterogeneity within the infected leaf. Albugo candida also caused localized changes in the carbohydrate metabolism of the leaf; soluble carbohydrates accumulated in the infected region whereas the amount of starch declined. There was an increase in the activity of invertases which was confined to regions of the leaf invaded by the fungal mycelium. The increase in apoplastic invertase activity was of host origin, as mRNA levels of the ATb FRUCT1 gene (measured by semiquantitative RT-PCR) increased 40-fold in the infected region. The increase in soluble invertase activity resulted from the appearance of a new isoform in the invaded region of the leaf. The resistant and moderately resistant cultivars contained higher amounts of chlorophyll, sugars and total phenols than the susceptible cultivar at all growth stages. However, total proteins and free amino acids were higher in the susceptible cultivar at all growth stages (Singh, 2000). Information on chromosome number and meiotic chromosome configuration is tabulated for 3 B. juncea lines developed at the Agriculture Canada Research Centre in Saskatoon: TO97-3360 (BC4F4) with high oleic acid content (68.6%), TO97-3400 (BC3F4) which is resistant, and TO973414 (BC3F4) has a low alkenyl glucosinolate content (28 micro moles/g defatted meal) (Cheng et al., 1999). Higher starch contents were found in noninfected tissues, and it is suggested that this could be due to the higher alpha amylase activity in diseased tissues (Debnath et al., 1998). Chlorophyll has a positive role in A. candida resistance in Indian mustard (Gupta et al., 1997). Thaumatin-like Journal of Oilseed Brassica, 5 (special) : Jan 2014 protein (PR-5), associated with the resistance of B. juncea towards A. candida which is not found previously. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell’s immune response (Kaur et al., 2011a). 9.2 RNA content In lpomoea WR there was greater reduction in the RNA content of infected tissues than in the healthy, adjacent tissues (Misra and Padhi, 1981). 9.3 Photosynthesis Black et al. (1968) used infrared CO2 analysis to demonstrate that a decline in the photosynthetic rate of cotyledons of radish infected with A. candida preceded the rise in respiration rate reported by Williams and Pound (1964). In another study, Harding et al. (1968) examined the pattem of pigment retention during green island development following infection of B. juncea cotyledons with A. candida. They found that labelled glycine 2-14C was incorporated into chlorophyll a and b in both infected and non-infected tissue. Both tissue fixed 14 CO2 in the light, but 4 days after infection green islands fixed five times more 14CO2 in the light than did noninfected tissue. The maintenance of chlorophyll and continued photosynthetic activity in green island tissue was parallaled by delayed breakdown of chloroplasts. Extensive research has indicated that the overall activity of photosynthetic pathways declines in leaves infected by rusts and PM, and is accompanied by a decrease in chlorophyll content of the tissue (Cooke, 1977; Daly, 1976). 9.4 Accumulation of metabolites Long et al. (1975) suggested that invertase may play a key role in the provision of substrate for the accumulation of starch at infection sites: where there is a surplus of soluble carbohydrate, particularly sucrose, hydrolysis by invertase might provide hexose for starch synthesis within chloroplasts. Invertase may thus mediate a system by which the excess soluble carbohydrate at infection sites is 9 converted to osmotically inactive polysaccharides. Dhingra et al. (1982) found decreased amounts of free protein, total protein and total phenolic compounds in floral parts and floral axes of B. rapa infected with WR. Dhawan et al (1981) correlated resistance of B. juncea cv. RC-781 with higher concentrations of phenols when compared with the susceptible cvs. Prakash and RH-30, where greater amount of sugar was present. Singh et al. (1980) demonstrated that cellulase, endo-PMG and endo-PG were produced in B. juncea leaves infected with A. candida. Maheshwari and Chaturvedi (1983) found that the swelling and disruption of subcellular particles rich in lysomal acid hydrolases was produced by acid phosphatase activity centered primarily in the infected tissues of B. juncea. Acid phosphatase activity in antheridia, oogonia and oospores of A. candida indicates that this enzyme plays a role in the synthesis of fungal organs. 9.5 Growth substances: Infection of host plants with Albugo causes hyperplasia and hypertrophy of leaf, stem and floral parts. Kiermayer (1958) found that these symptoms are produced in plants infected with A. candida due to the production of indolacetic acid (IAA). Hirata (1954, 1956) found that infection with A. candida causes an initial increase in diffusible auxin in diseased stems and leaf sections, followed by a decrease before maximum development of the galls. The auxins in healthy and Abugo-infected inflorescences of B. napus have now been identified and estimated quantitatively by Srivastava et al. (1962). Malformed B. napus inflorescences produce IAA, IAN, accelerator L, and an etherinsoluble growth substance designated as A. Kumari et al. (1970), and Lal et al. (1980) studied the quantitative and qualitative changes in the amino acid contents of diseased (hypertrophied) and healthy tissues of mustard and radish. The infection causes the breakdown of plant proteins, releasing small quantities of tryptophan, which reacts with endogenic phenolic acid to produce IAA which is responsible for hypertrophied growth. It was possible to recover waxy or medium waxy B. juncea types with WR 10 Journal of Oilseed Brassica, 5 (special) : Jan 2014 resistance, though in low frequencies (Subudhi and Raut, 1994). More research is needed to gather basic information concerning the effects of WR on respiration, photosynthesis, accumulation and transfer of carbohydrates, production of growth regulators, and the role of phenolics and other growth substances in infected host tissues. 9.6 Plant defense resistance genes Plant defenses against colonization by a pathogen thought to be triggered by either direct, or indirect interaction between proteins encoded by the pathogen avirulence (Avr) gene and a corresponding plant resistance gene. From previous molecular genetic analyses of downy mildew resistance, there are numerous examples of receptor-like genes in A. thaliana that vary in different modes of defense regulation (Eulgem et al., 2004; Holub, 2001; McDowell et al., 2000; Tör et al., 2002). The majority of plant R genes encode nucleotide-binding site leucine-rich repeat (NB-LRR)-type proteins which can be further grouped into two subclasses based on their N-terminal sequence: those containing a coiled-coil (CC) domain (CC-NB-LRR), or those containing a domain with similarity to Drosophila toll and mammalian interleukin-1 receptor (TIR) (TIR-NB-LRR) (Hammond-Kosack and Jones, 1997; Jones and Jones, 1997; Young, 2000). LRR is involved in protein–protein interactions and occur in a number of proteins with different function (Kobe and Deisenhofer, 1994, 1995). Domain exchange between LRR of closely related R genes supports their role in pathogen recognition (Ellis et al., 1999; Wulff et al., 2001). Variation among R-genes occurs mainly in their LRR domain, typically in the solvent exposed â-strand/â-turn structure within the LRR domain. Based on their similarity with some of the animal proteins involved in apoptosis and innate immunity, The N-terminal domain of plant R-proteins are thought to have to function as a signaling domain (Rairdan and Moffet, 2007). However, several recent reports indicate that the N-terminal domains of NB-LRR proteins may be involved in recognition specificity (Moffett, 2009). The high variability of LRR domains and their role in protein-protein intreraction led to the idea that R-proteins interact directly with their congnate Avr proteins. However there is a limited evidence for such a direct intreation which led to the development of Guard and Decoy hypotheses which propose that R-proteins detect interaction of Avrs with host proteins (Dangl and Jones, 2001; van der Hoorn and Kamoun, 2008). Recognition of a pathogen by a plant initiates a rapid response localized to the infection site and manifested by changes in ion flux and production of reactive oxygen species that lead to induction of downstream signals and defense genes (Kombrink and Schmelzer, 2001; Morel and Dangl, 1997). Initiation of local defense also results in signals that induce systemic acquired resistance (SAR) in noninfected distal parts of the plant, resulting in broadspectrum resistance (Dong, 2001; Shah and Klessig, 1999). The role of salicylic acid (SA) in plant defense and induction of SAR has been shown by treatment of plants with SA or its synthetic analogs such as 2, 6-dichloroisonicotinic acid (INA) and benzothiadiazole (Klessig et al., 1994). Furthermore, transgenic plants expressing the bacterial SAdegrading enzyme, NahG, are unable to induce SAR (Delaney et al., 1995). Several mutants in A. thaliana have been identified that affect disease resistance responses associated with defense regulatory genes such as: AtSGT1b (homolog of the yeast gene SGT1) (Austin et al., 2002; Tör et al., 2002), EDS1 (enhanced disease susceptibility) (Parker et al. 1996), NDR1 (non-racespecific disease resistance) (Century et al., 1997), PAD4 (phytoalexin deficient) (Glazebrook et al., 1997), and RAR1 (homolog of a barley gene required for Mla powdery mildew resistance) (Muskett et al., 2002; Tornero et al., 2002). Resistance specified by the RPS4 gene to the bacterial pathogen Pseudomonas syringae expressing avrRps4 (Gassmann et al., 1999), and the oomycete Peronospora parasitica specified by RPP1, RPP2, RPP4, and RPP5, which all encode TIR-NB-LRR proteins, is abolished by eds1 (Aarts et al., 1998; Parker et al., 1996; Rusterucci et al., 2001). White rust in natural populations of A. thaliana has Journal of Oilseed Brassica, 5 (special) : Jan 2014 been attributed to distinct Albugo species, A. candida and A. laibachii (Thines et al. 2009). Three resistance genes to A.candida (recently renamed as A. laibachii; Kemen et al., 2011) (RAC) isolate Acem1 were identified (Borhan et al., 2001). Cloning was reported of the first WR resistance gene to isolate Acem1 of A. candida (RAC1) from Ksk-1 accession of A. thaliana. They also describe the effect on RAC-mediated resistance of standard mutations that previously were used to characterize defense signaling in DM resistance. RAC1 is a member of the Drosophila toll and mammalian interleukin-1 receptor (TIR) nucleotidebinding site leucine-richrepeat (NB-LRR) class of plant resistance genes. Strong identity of the TIR and NB domains was observed between the predicted proteins encoded by the Ksk-1 allele and the allele from an Acem1-susceptible accession Columbia (Col) (99 and 98 %, respectively). However, major differences between the two predicted proteins occur within the LRR domain and mainly are confined to the â-strand/â-turn structure of the LRR. Both proteins contain 14 imperfect repeats. RAC1-mediated resistance was analyzed further using mutations in defense regulation, including: pad4-1, eds1-1, and NahG, in the presence of the RAC1 allele from Ksk-1. White rust resistance was completely abolished by eds1-1, but was not affected by either pad4-1 or NahG (Borhan, 2004). A second white rust resistance gene (WRR) named WRR4 was also cloned form A. thaliana accession Col (Borhan et al., 2008). WRR4 encodes for a TIR-NB-LRR protein and is resistance to A. candida races from Brassica (races 2, 7 and 9 from B. juncea, B. rapa and B. oleracea respectively) as well as race 4 from Capsella bursa-pastoris. WRR4 resistance is dependent on the functional expression of eds1. Expression of the A. thaliana WRR4 in B. juncea (susceptible to race 2) and a B. napus lines susceptible to race 7, provided full immunity (Borhan et al., 2010). A single gene (Acr) responsible for conferring resistance to A. candida was mapped on a densely populated B. juncea RFLP map. Two closely linked 11 RFLP markers identified (X42 and X83) were 2.3 and 4 cM from the Acr locus, respectively (Cheung et al., 1998). 9.7 Albugo Genome Genetic and genomics research on Albugo was hampeered by the fact that it is an obligate biotroph pathogen and could not be cultured axenically. However recent technological advances in genome sequencing and advent of next generation sequencing technology has made it possible to sequence the genome of two Albugo species, A. candida (Links et al., 2011), and A. laibachii (Kemen et al., 2011). Albugo candida has a compact geneome (approximately 45 Mb) and it is almost half of the the oomycete Hyaloperonospora arabidopsidis genome (99Mb). Another feature of A. candida geneome is reduction in the number of pathogenicity factors and reduced retention of certain bosynthetic pathways, fatures that are for A. laibachii and are signature of biotrophy. 10. Techniques 10.1 Growth chamber inoculation technique In a most widely used growth chamber inoculation technique Verma et al. (1975) and Verma and Petrie (1979) described that seeds of the susceptible Brassica cultivar are planted 2 cm deep in a soilfree growth medium (Stringham, 1971) in 10 cm square plastic pots. Seedlings are thinned to ten plants per pot. Plants are grown in a growth chamber with an 18-h photoperiod (312µEM-2S-1) and at day-night temperatures of 21oC and 16oC, respectively. Pots are placed in metal trays and watered by flooding the trays. Inoculum was prepared by dispersing zoosporangia from pustules from-infected fresh or frozen leaves in deionized distilled water, filtered through cheese cloth, germinated for 2-3h at 5oC, and adjusted to 75000-100,000 zoopores per ml. The inoculum was sprayed on to plants with an atomizer until leaf runoff. Control plants were sprayed with distilled water. The plants were placed in a mist chamber (100 % relative humidity) in the growth chamber for 72 h at 16oC to promote infection, and disease incidence and severity recorded 10-days after inoculation. Several greenhouse and growth 12 Journal of Oilseed Brassica, 5 (special) : Jan 2014 chamber inoculation techniques with similar parameters have also been reported (Goyal et al., 1996 b; Singh et al., 1999; Bansal et al., 2005). Rimmer et al. (2000), and Li et al. (2007) used WR pustules collected 10 days prior to inoculation (dpi) and stored at -80oC. For use as an inoculum, zoosporangia were dispersed from infected cotyledons into deionized water and filtered through cheesecloth to remove plant debris. The concentration of the zoosporangia was determined using a haemocytometer and adjusted to 105 zoosporangia ml_1. Fully expanded cotyledons from seedlings, 10 days after sowing, were inoculated by spotting 10µl of the zoosporangial suspension onto the adaxial surface of each of the two lobes of each cotyledon. Plants were then subjected to 4 days of enhanced humidity (=95 % RH). At 4–5 leaf stage (4 weeks after sowing when the 5th leaf was emerging), plants were inoculated by spraying a suspension of 10 5zoosporangia/ml until run-off. Plants were subjected to 4 days of enhanced humidity by placing each pot into a sealed plastic bag that had been pre-moistened with DI water. The propagators were placed in an air-flow-bench under spore free conditions in the glasshouse at 18°C ± 2°C (Jenkyn et al, 1973; Nashaat & Rawlinson, 1994) with supplementary light to maintain a 16 h light/ 8h dark; day/ night cycle. The seedlings were sprayed with sterilised distilled water (SDW) to clean the surface of cotyledons 24 h prior to inoculation. Meena (2007) prepared sporangial suspension by adding 1 to 2 ml SDW to glass vial containing excised frozen or freshly sporulating cotyledons. The vial was shaken vigorously on a vortex shaker to facilitate the release of sporangia from the sporangiophores. The concentration of the sporangia was determined using haemocytometer and adjusted to 2.5 x 10 4 sporangia/ml. Each cotyledon was inoculated with two 5ml droplets of sporangial suspension using a micropipette. Alternatively, the plants were sprayed to run off with the spore suspension using an atomiser. After inoculation, the propagators were covered with clear plastic lids and sealed with insulation tape to maintain approximately 100% RH. The plants were then placed in a growth chamber for 12 days at 16ºC with 8 hours darkness initially, followed by 16 hours photoperiod with 70-120µmol/m2/s irradiance. 10.2 Oospore germination The most conspicuous symptoms of WR and probably the major cause of yield loss are distortion and hypertrophy of infected inflorescence called “staghead”. When ripe, stagheads are almost entirely composed of numerous brown, thick-walled oospores, the form in which the pathogen survives during the off-season, and also the source of primary infection. Despite their importance in the epidemiology, conditions under which the oospores germinate have largely been a mystery until the reports of Petrie and Verma (1974) and Verma and Petrie (1975). Prior to this report, only De Bary (1866) and Vanterpool (1959) have described oospore germination in A. candida. Vanterpool (1959) reported germination as “always irregular and uncertain”, never exceeding 4 % of the spores. Verma and Bhowmik (1988) observed that treatment of oospore with 200ppm KMNO4 for 10 minutes induces increased germination. Petrie and Verma (1974); Verma and Petrie (1975), however devised three reproducible techniques which all gave very high percentage of germination. In the first method, a small amount of finely ground staghead powder consisting largely of oospores was scattered over moist filter paper placed on wet cotton in a petri dish; the lid of the dish was also lined with moist cotton. The plates were incubated at 10-15oC for the period of up to 3 weeks. In the second method, sterile deionized water or strile or non-strile tap water was allowed to drip slowly onto sintered glass filters of ultrafine porosity where small amounts of oospore powder were scattered. This was done in an attempt to mimic the leaching action that might occur during spring from melting snow or rain. Most of these experiments were run at 10-15oC. In the third method, which the authors most routinely used, a small amount of oospore powder was placed in 50 ml sterile water in a 125 ml flask and incubated at 200 rpm on a rotary shaker at 18-20 oC for a period of 3-4 days. The spore suspension was then poured into a petri dish and kept stationary at 13oC for 24h or more. Counts of germinated oospore were made on materials mounted in lactophenol-aniline blue. Journal of Oilseed Brassica, 5 (special) : Jan 2014 All three techniques induced germination of oospores in large numbers. Washing of oospores on a rotary shaker for 3-4 days followed by a day in still culture was the most rapid method and gave the highest percentage germination. Oospores required 2 weeks of washing on a sintered glass filter before maximum germination was obtained. On moist filter paper, maximum germination occurred after an incubation period of 21 days. Three distinct types of germination were observed. In the most common type, the oospore content was divided into numerous zoospores which were then extruded into a globular, thin-walled sessile vesicle. Zoospores subsequently escaped from the vesicle. Initiation of a vesicle to zoospore escape was completed in 3.0-5.2 minutes with an average elapsed time of 4.1 minutes. Between 40 and 60 zoopsores were formed per vesicle (Verma and Petrie, 1975). 13 10.3 Oospores as primary source of inoculum Oospores are formed in the A. candida-infected hypertrophied tissues of inflorescence, stem, pod, roots (Lakra and Saharan, 1989b; Goyal et al., 1996b) and senesced leaves (Verma and Petrie, 1978). The oospores are important both for initation of the disease (Butler, 1918; Butler and Jones, 1961; Chupp, 1925; Kadow and Anderson, 1940; Walker, 1957), as well as for the survival of the pathogen in the absence of the host (Verma and Petrie, 1975). In the second germination type, observed only infrequently, a germ tube was produced from the germinating oospore and zoospores which were differentiated in the oospore were discharged through the tube into a so-called “terminal vesicle” formed at the end of the tube. Zoospores subsequently escaped from the vesicle (Verma and Petrie, 1975). A less commonly observed mode of germination was by a simple or branched germ tubes. Occasionally up to three branches were observed on a germ tube (Verma and Petrie, 1975). However, in the absence of a reliable method of germination, the role of oospores both as overwintering agent, as well as incitant of primary infection have largely been speculated. Even after the germination of the oospores, information is still locking whether the zoospores from germinating oospores are capable of infecting rapeseed host plants. Since the emerging cotyledons are the most likely infection sites in the field, Verma et al. (1975) grew plants of susceptible B. rapa cv. Torch in the growth chamber (under conditions described earlier) kept them at cotyledon stage by removing the growing points. Cotyledons of 10-day-old plants were drop-inoculated with zoospore suspension derived from germinating oospores. Plants were kept under a mist for 3 days. Ten days after inoculation nearly every inoculated plant showed heavy infection in the form of white pustules on the underside of cotyledons. These infection studies suggest that zoospores from germinating oospore are the main infecting units for initiation of primary infection. We still do not know how long oospore can remain viable in soil or plant debris. In their extensive studies on viability of oospores, Verma and Petrie (1975) however, reported that more than 50% of the oospores germinated in all staghead samples with the exception of 1953, 1956 and 1959. Germination of 43 % of oospores from staghead material kept in storage for 20 years (1953 material) does indicated their potential longevity. Since the authors recorded the highest percentage of germination (70 %) in 1973 samples which had been collected only 2 weeks prior to the test, their results suggest that oospores do not appear to require any dormancy period. Verma and Petrie (1980) also investigated the importance of oospore as a source of primary inoculum in a field experiment conducted under irrigated and dry land conditions. The treated plots were seeded with seeds of susceptible B. rapa cv. Torch mixed with an equal weight of oospore powder. The control plots received no oospore powder. Both number of pustules per infected leaf, and the percentage of plants with stagheads were significantly higher in oospore-infested than those in the non-infested plots. These results convincingly suggest that oospores over-wintered in soil, or carried on the seed as contaminant, are most likely the primary source of infection. 14 Journal of Oilseed Brassica, 5 (special) : Jan 2014 Recently, Meena and Sharma (2012) used a mixture of 1 % b-glucuronidase arylsulfatase (available from Sigma) for germination of oospores from hypertrophied plant tissues in 1:9 ratio in SDW which was then stored in 10ml vials in the refrigerator. 100 mg of staghead powder was suspended in 10 ml enzyme dilution and incubated on a rotary shaker (200 rpm) at room temperature for 24 h. On the second day the suspension was centrifuged to pellet the spores and washed three times with 20 ml SDW (mixing them by centrifuging after each wash). The oospore suspension was returned to the rotary shaker for 48-72 h, centrifuged, resuspended in fresh water daily till the sixth day, suspension transferred to empty flask, and chilled at 10ºC for 24 h. The suspension was removed from refrigerator and brought to room temperature before inoculation. 10.4 Induction of staghead in flower-bud inoculated plants In the past, it was a common blief that the hypertrophies or stagheads are produced as a result of early infection of young seedlings and systemic development of the fungus in the plant. However, this theory was rejected when Verma and Petrie (1979) and Goyal et al. (1996b) routinely obtained stagheads by artificially inoculating flower buds of plants grown under growth chamber and greenhouse conditions. These results of growth chamber and of several field experiments (Verma and Petrie, 1979, 1980) conclusively proved that a large percentages of stagheads in the field are produced as a result of secondary infection of flower buds rather than a systemic development of the fungus in the plant. This flower bud inoculation technique at growth stage 3.1 (Goyal et al., 1996b) is now routinely being used for screening advanced breeding lines at the Agriculture Canada Research Station, Saskatoon, Canada. Results of these studies are also useful in determining actual time of application of both protectant and systemic fungicides to control WR. 10.5 Detached-leaf culture technique In order to make more economic use of growth chamber space for screening germplasm for resistance, and to determine effects of abiotic factors on temporal development of A. candida infection and oospores development, Verma and Petrie (1978) investigated use of detached-leafculture-technique. Healthy leaves from the rosette of 12-14-day-old B. rapa seedlings are detached and transferred to petri dishes containing 20-25 ml of autoclaved medium consisting of 0.5 ppm benzyl adenine and 0.8% agar. Leaves are placed in the dishes with their lower surface on the medium usually within 15 minutes of detachment. Four leaves are placed in a plate and at least 20 leaves are used per treatment. Leaves are drop-inoculated with a zoospore suspension (75,000-100,000 zoospore/ml) derived from zoosporangia of A. candida race-7. Control leaves are treated with distilled water. A clean but generally non-sterile technique is used and no attempt is made to manipulate leaves aseptically or to sterilize the inoculum. Leaves are kept under 100 % relative humidity for 72-h with day-night temperatures of 21 and 16 o C, respectively. Following an initial 24-h dark period, an 18-h day (312µEM -2S-1) is maintained for the duration of the experiment. Observations are recorded 14 days after inoculation. Plant susceptibility ratings of various Brassica species and breeding lines on the inoculated detached leaves are essentially the same as when intact plants are used as the host. In addition, the detached-leafculture-technique has several advantages to the researchers. The method facilitates the establishment and maintenance of single zoospore cultures and should enable almost complete isolation from extraneous inoculum, including races of A. candida. Detached-leaf culture also results in greater uniformity of experimental units, more economic use of growth and mist chamber space, and allows greater use of environmental control. From the plant breeder’s point of view, the program efficiency is increased, since the breeder can select resistant material for inter-crossing from among a vigorous growing plant population rather than a weak group of resistant plants that have survived the unfavourable environment necessary to obtain differential infection on potted plants. 10.6 In vitro callus cultures of A. candida Preliminary dual in-vitro-culture of A. ipomoeae- Journal of Oilseed Brassica, 5 (special) : Jan 2014 panduraneae and species of Ipomoea (Singh, 1966), A. candida race 2 and B. juncea (Lahiri and Bhowmik, 1993), and unidentified race of A. candida and B. juncea (Goyal et al., 1995) have been established. Although, investigators report the presence of zoosporangia and oospores in callus tissues derived from hypertrophied stems (Singh, 1966, Goyal et al., 1995), or hypertrophied peduncles or thickened terminal leaves (Lahiri and Bhowmik, 1993), but the origin of both sexual and asexual spores is questionable, because the hypertrophied tissues used as explants in their studies are known to almost entirely composed of thick-walled oospores (Verma and Petrie, 1975; 1979; Saharan and Verma, 1992; Verma and Bhowmik, 1988). Using explants from freshly-inoculated leaves, Goyal et al., (1996c) very successfully established dual-in vitro callus cultures of A. candida race 7V and B. rapa cv. Torch on MS medium (Murshige and Skoog, 1962) supplemented with 1.0mgL-1 ´naphthalene acetic acid and 1.0 mgL-1 benxylaminopurine. These authors have provided evidence for: a) production of zoosporangia, oospores, and parthenogenetic-like oospores; b) establishment of haustorial-connections with host cells; c) origin and development of both antheridia and oogonia; and d) the pathogenicity of the zoospores from in-vitroproduced zoosporangia and oospores. Goyal et al. (1996c) reported that : a) callogenesis was observed within 7-8 days of incubation; b) proportion of callused explants was significantly affected by the type and concentration of growth regulators; c) under both light and dark conditions, the length of incubation period significantly affected the presence and development of haustoria, zoosporangia, oogonia, antheridia and oospores; and d) the callus tissues incubated in the light were hard, nodular, and green, compared-to soft, watery, and become yellow in the dark. Zoosporangia were observed in the longest numbers of calli at 8 days incubation, and after this their numbers declined consistently; zoosporangiophores without zoosporangia grew out of the callus cells after 18 days of incubation. In callus cells, the zoosporangiophores were long, 15 knotted, branched, and indeterminate, compared to the short, club-shaped, unbranched, and determinate in infected leaves. By subculturing the calli every two weeks, for 18 weeks, the A. candida- B. rapa dual cultures were maintained. After 18 days of incubation and until the end of the observation period, haustoria similar to those reported in infected leaf tissues (Verma et al., 1975) were observed in the cytoplasm of callus cells, or between the cell wall and the cell membrane. The development of antheridia and oogonia among the callus cells were observed after 13-days of incubation and until the end of the observation period. Two types of oospores, mature oospores with characteristic features including wall layers and a coenocentrum, or two coenocentra, and parthenogenetic-like oospores were observed after 18-days of incubation. The parthenogenetic-like oospores were oval, devoid of warty layers like typical mature oospores, often germinated by a germ tube, and were associated with haustoria inside the callus cells. Pathogenecity test on seedlings of B. rapa cv. Torch using zoospores derived from in-vitro-produced zoosporangia and germinating oospores confirmed the viability and the virulence of A. candida in dual callus cultures (Goyal et al., 1996c). The A. candida –B. rapa dual culture system reported by these authors has potential for sexual studies of the fungus. Because it was possible to trace the development of antheridia and oogonia from the mycelium, which support the view that isolates of A. candida race 7V are homothallic. This dual culture system can also be useful in vitro selection studies for recovering resistant cells. Debnath et al., (2001) reported that the host callus and the pathogen establishes a complete balance in culture, and the morphology of the mycelium, haustoria, zoosporangia, antheridia, oogonia and oospores in dual culture is identical to that of infected intact plant. Oospore formation is favoured over that of sporangia, and oospore germination by germ-tube is evident. Growth of dual culture is influenced by light quality, temperature, vitamins, carbohydrates and amino acids in the medium. These 16 Journal of Oilseed Brassica, 5 (special) : Jan 2014 differential responses can be used for future studies on host pathogen interactions and for breeding of disease resistant plants. Lahiri and Bhowmik (1993) maintained A. candida in infected callus tissue for prolonged periods by periodic subculturing and it kept pace with the growth of the callus tissue. 11. Epidemiology Temperature gradient plates (Smith and Reiter, 1974) and detached-leaf-culture technique (Verma and Petrie, 1978) were used to determine effect of temperature on temporal progression of white rust on a) leaves of different ages, b) leaves detached at the end of light and dark periods, c) type and number of zoosporangial pustules on abaxial and adaxial leaf surfaces, and development of oospores (Verma et el., 1983; Goyal et al., 1996a; Bartaria and Verma, 2001). It is essential to determine exact parameters for disease development before detached-leaf-culture technique can be used to screen rapeseed-mustard cultivars for resistance against A. candida. 11.1 Temperature effects on disease development: Temperature, leaf age, time of leaf detachment, and the interaction of these factors had a significant effect on the temporal development of A. candida race 7 on detached leaves (Verma et al., 1983). Of the temperatures tested (3-32oC), 21oC gave the best disease development, with 18.5 oC being the calculated optimum. The disease did not develop at 3o, 29o, and 32o C, and was slow to develop at 9o, 12 o, and 27oC. There was a highly significant (p<0.01) interaction between length of incubation period and temperature. Unlike intact plants, detached leaves developed pustules on both surfaces. Infection occurred on leaves of all ages, but medium-aged leaves supported the maximum number of pustules, followed by the younger leaves. Leaves detached at the end of a dark period developed more pustules than those detached at the end of light period. While using detached leaf culture technique for screening germplasm for resistance to white rust, Verma et al. (1983) advised inoculation of adaxial surface of cotyledons of medium aged leaves, with an incubation temperature of 18-22oC. Sullivan et al. (2002) observed that only 3 h of leaf wetness is required for disease development at optimum temperature range of 12 to 22°C. The nonavailability of forecast system for major diseases of oilseed Brassicas in India does not allow farmers to make timely and effective fungicidal sprays. In one of the multilocation study conducted for 8 years, Chattopadhyay et al. (2011) observed the initiation of WR disease on leaves of mustard during 29-131 days after sowing (DAS), highest being at 54 DAS. Severity of WR disease is favoured by >40 % minimum afternoon and >97 % maximum morning relative humidity (RH) and 16-24 o C maximum temperature. Staghead formation is significantly and positively influenced by 20-30 o C maximum (>12 o C minimum) temperature and >97 % maximum morning RH. 11.2 Temperature effects on oospore development Epidemiological studies on A. candida have focused on the production, viability and germination of zoosporangia (Melhus, 1911; Endo and Linn, 1960; Lakra et. al., 1989), and the influence of host age and time of leaf detachment on development of the disease (Verma et. al., 1983). Little is known about the sexual reproduction and genetics of the fungus due to the difficulty in determining the factors responsible for induction of the sexual reproductive phase. The effect of temperature on in vitro germination of oospore has been reported (Verma and Petrie, 1975), however, information on the optimum temperature and the time required for production of oogonia, antheridia and mature oospore in leaf tissue would assist in designing experiments for the study of oogenesis, fertilization and karyogamy. Using temperature gradient plate (Smith and Reiter, 1974) and detached leaf culture technique (Goyal et al., 1996a) established effect of temperature and incubation period on progressive development of oospores of A. candida race 2V in B. juncea leaves. The progressive development of A. candia oospores in detached leaves of B. juncea is largely Journal of Oilseed Brassica, 5 (special) : Jan 2014 dependent on incubation temperature. Oogonia and oospore production occurred over the entire range of incubation temperatures of 10-27oC. The earliest development of oogonia is observed at 25oC, 7days after inoculation and incubation. The largest number of oogonia at the 21o, 23o, 24o and 25oC treatments is observed 12 days post inoculation and numbers decreased thereafter; at lower and higher temperatures; development of oogonia occurred later. Maximum numbers of oogonia are recorded after 17 days at 15oC treatment at the end of the experiments. Mature oospores are observed 12 days after incubation at 23o and 24oC. The number of mature oospores was still increasing at 17 days postinoculation in all treatments. Mature oospores developed later and more slowly at lower and higher incubation temperatures. The production of A. candida oospores in leaf tissues can be important in disease perpetuation. Hypertrophied tissues (staghead) are quite resistant to decomposition and the release of oospores can take 3-4 years. Leaf tissues are quick to decompose, and thus oospore release from such material could be expected the following year. In naturally-infected leaves, oospores are produced in the later part of the season when temperatures are warm (Verma, 1989). Warm temperatures hasten leaf senescence, which in turn enhances tissue decomposition and early release of oospores. The knowledge of an optimum temperature and time for the development of oospores in detached leaves in their study made it possible to compare the sequential events of oogenesis, fertilization and karyogamy in various Albugo species at the earliest stages of their development. These comparative investigations in Albugo species can also be useful in fungal taxonomy. The detached leaf culture technique for oospore development can also be used to determine the heterothallic nature of A. candida. 11.3 Temporal development of A. candida infection in cotyledons: Verma et al., (1975) determinied temporal progression of WR infection in cotyledons of susceptible (B. rapa, B. juncea), moderately 17 resistant (B. hirta), and immune (B. napus) cultivars. Cotyledons of all four Brassica species were inoculated with zoospores of A. candida produced from germinating oospores or zoosporangia. At different times after inoculation, whole cotyledons were fixed in 95 % ethanolacetic acid (v/v) solution, cleaned in 70 % lactic acid at 40oC for 3-4-days, and stained with cotton blue in lactophenol. The preparation was examined under the compound microscope. Generally, the sequence of events from zoospore encystment to formation of the first haustorium was the same in all hosts, although under field conditions, Brassica hirta is moderately resistant and B. napus is essentially “immune”. In B. juncea the first haustorium was observed 16-18 h after inoculation, while in B. rapa, B. hirta and B. napus the first haustorium was observed about 48 h after inoculation. In the susceptible hosts, after the formation of the first haustorium, the hyphae grew rapidly and produce variable number of haustoria in each cell. The profusely branched, nonseptate mycelium appeared to fill all available intercellular spaces, and in five to six days after inoculation, the club-shaped zoosporangia develop from a dense layer of mycelium. In the immune host, usually only one haustorium was formed, after which the hyphae ceased to elongate. At about 72 h after inoculation, a fairly thick, densely stained encapsulation was usually detected around each haustorium, and later only “ghost” outlines of hyphae and haustoria were observed. Encapsulations were not observed around haustoria of susceptible hosts. From these observations (Verma et. al., 1975) it seems probable that zoospores derived from germinating oospores constitute the primary inoculum for infection of cotyledons of susceptible Brassica species. No evidence of direct infection by the germ tubes was seen (Verma and Petrie, 1975). The establishment and maintenance of compatible relationship between A. candida and its hosts hinges on the successful formation of the first haustorium. A similar sequence of events in both susceptible and immune hosts upto this point suggests that there 18 Journal of Oilseed Brassica, 5 (special) : Jan 2014 appears to be no morphological barrier to zoospore encystment, germination and subsequent penetration through stomata. In the incompatible combination it is not clear whether the parasite fails to produce a functional haustorium, or whether a viable haustorium is formed within the host cell and is subsequently killed by the host’s defence mechanism. The fairly dense, thick encapsulation observed around haustorium of immune host tissue suggests that the later may be the case. In any event it does seem that the decision between compatibility and incompatibility is made within 48 h after inoculation. Studies using whole mounts (Verma et.al., 1975) can provide a rapid and useful quantitative means of measuring fungal development and can be useful in screening for disease resistance or testing the effects of environmental changes or fungicide treatments. Whole mounts may also provide a useful perspective for ultrastructural studies where the total amount of fungal thallus present in a susceptible host is not always appreciated. Certainly, the massive amount of intercellular mycelium, particularly the much-branched sporangiophore “base”, which the host is capable of supporting while still actively photosynthesizing, emphasizes the highly integrated and delicate control occurring in the type of parasitism that has evolved in A. candia. 12. Association of Albugo and Hyaloperonospora The association or mixed infection, or simultaneous occurance of A. candida and Hyaloperonospora brassicae pathogens on leaves, inflorescence and silique of oilseed Brassica in nature is very common (Saharan and Verma, 1992). The intensity of mixed infections varies from 0.5 to 35.0 per cent. It is reported that A. candida predisposes the host tissues to infection by H. brassicae (Bains and Jhooty, 1985; Saharan and Verma, 1992; Saharan and Mehta, 2002). However, Soylu et al. (2003) reported that the H. parasitica infections are first apparent to the naked eye as a carpet or ‘‘down’’ of conidiophores covering the upper and lower surfaces of leaves and petioles, a symptom characteristic of DM diseases. The zoosporangia of H. parasitica emerge in profusion from stomata without forcible damage of host tissue (Borhan et al., 2001). While both these pathogens usually exist as specialized pathotypes on different cruciferous species, and even on different cultivars within a species, asexual reproduction, in general, is most prolific on the particular host of origin (Mathur et al., 1995a; Nashaat and Awasthi, 1995; Petrie, 1988; Pidskalny and Rimmer, 1985; Saharan and Verma, 1992; Silue et al., 1996). Normally A. candida occurs in intimate association with H. parasitica (Holub et al., 1991) including on stagheads (Awasthi et al., 1997) in crucifers. Bains and Jhooty (1985) found that H. parasitica colonies commonly occurring among those of A. candida on plant tissues. They studied the association of H. parasitica with A. candida on B. juncea leaves and proposed that A. candida biochemically pre-disposes the host plants to H. parasitica. However, because the incubation period of H. parasitica is shorter than that of A. candida, they found that H. parasitica colonies tend to develop first, followed by A. candida under glasshouse conditions. In contrast, they found that the situation could be the reverse under natural conditions (Bains and Jhooty, 1985). Under field conditions, A. candida possesses the capacity to elevate the incidence and severity of infection by H. parasitica in crucifers (Constantinescu and Fatehi, 2002), and similar situations have been described for H. arabidopsis in Arabidopsis thaliana (Holub et al., 1991) and in B. juncea (Cooper et al., 2008) after pre-inoculation with A. candida. Singh et al. (2002a) studied that the infection of B. juncea with a virulent isolate of H. parasitica inhibited or adversely affected the development of a virulent isolate of A. candida after simultaneous coinoculation of B. juncea, while an avirulent isolate of A. candida induced host resistance toward H. parasitica. Previous findings suggest that the inoculation order of the two pathogens may be a critical factor in determining the outcome of the interaction of two pathogens. Kaur et al. (2011b) observed that the inoculation of B. juncea with an asymptomatic isolate of H. parasitica and subsequently with a virulent isolate of A. candida, not only reduced the incubation period but also increased the severity of disease caused by the WR pathogen. They also Journal of Oilseed Brassica, 5 (special) : Jan 2014 determined that although H. parasitica was asymptomatic in the host, it systemically colonized host tissues away from the site of inoculations. 13. Pathogenic variability in A. candida Physiological specialization has long been known in A. candida. Eberhardt (1904) recognized two specialized groupings of Albugo one attacking Capsella, Lepidium and Arabis, and other attacking Brassica, Sinapis and Diplotaxis; he was however, hesitant to use the phrase biological forms. Melhus (1911) also suggested the existence of specialization in A. candida. Pape and Rabbas (1920) demonstrated that the fungus on Capsella bursa-pastoris should be considered a distinct form. Savulescu and Rayss (1930) distinguished eight morphological forms within A. candida, and in 1946, Savulescu estabilished 10 varieties of A. candida based on host specialization and morphology. Hiura (1930) distinguished three biologic forms of A. candida on Raphanus sativus, B. juncea and B. rapa sp. chinensis. Napper (1933) described 20 races of A. candida in Britain. Togashi and Shibasaki (1934) found that sporangia of Albugo from Brassica and Raphanus were 20 x 18 µm in size, while those from Cardamine, Capsella, Draba and Arabis measured 15.5 x 14.5 µm, and classified these as macrospora and microspora, respectively. Results of these two Japanese studies (Hiura, 1930; Togashi and Shibaskaki, 1934) suggested that five distinct biological forms of Albugo were present. Subsequently, Ito and Tokunaga (1935) elevated the forms with larger spores to the rank of the species A. macrospora (Togashu) Ito. Biga (1955) recognized two morphological texa: A. candida macrospora and A. candida microspora, as proposed by Togashi and Shibaskaki (1934), but renamed them A. candida microspora and A. candida candida, respectively. On the basis of conidial measurements from 63 host species, Biga (1955) reported that A. candida microspora (15-17.5 µm diam.) was restricted to Armoracia, Brassica, Erucastrum, Raphanus and Rapistrum, whereas A. candida candida (12.5-15 µm diam.) had a wide range of cruciferous hosts. Endo and Linn (1960) reported a race of Albugo on Armoracia rusticana. 19 It is clear that each of the above authors were hesitant in describing specialized races of A. candida. Pound and Williams (1963) identified six races of A. candida: race I from Raphanus sativus var. Early Scarlet Globe; race 2 from B. juncea var Southern Giant Curled; race 3 from Armoracia rusticana var Common; race 4 from Capsella bursa-pastoris; race 5 from Sisymbrium officinale, and race 6 from Rorippa islandica. Verma et al (1975) and Delwiche and Williams (1977) added race 7 from B. rapa Turnip or Polish rapeseed and race 8 from B. nigra, respectively. Novotel’nova (1968) from USSR while analyzing intra-specific texa, reported that A. candida species consisted of separate morphological specialized forms confined to a particular range of host plants. Within the morphological forms, races can be differentiated, while within heterogeneous populations, both races and forms can be differentiated. It was considered that geographic and climatic conditions leave their distinguishing mark on the processes of form and populations of the fungus encountered by investigators from different countries. Novotel’nova and Minasyan (1970) and Burdyukova (1980) studied the biology of A. candida and A. tragopogonis in former USSR and conducted an in-depth study on the extent of specialization of A. candida. In India, Singh and Bhardwaj (1984) tested 12 Brassica species and identified 9 races from four hosts, viz, B. juncea, B. rapa var. Toria, B. campestris var Bbrown Sarson and B. rapa var. Pekinensis. Lakra and Saharan (1988c) identified five races of A. candida on the basis of its reaction on a set of 16 host differentials. They identified two distinct races from B. juncea which were different from the previous records. One (race 2), attacked B. nigra, B. juncea and B. rapa var. Brown Sarson, and the other (race 3) infected only B. juncea and B. rapa var Toria. Bhardwaj and Sud (1988) tested 26 cultivated and wild cruciferous hosts and identified nine new biological races from nine hosts, viz, B. rapa var. Brown Sarson cv. BSH 1, B. rapa var. Toria cv. OK-I, B. juncea cv. Varuna, B. chinensis. B. rapa var. Pekinensis cv. Local, B. rapa cv. PTWG, 20 Journal of Oilseed Brassica, 5 (special) : Jan 2014 Raphanus sativus cv. Chineses Pink, Raphanus raphanistrum wild radish and Lepidium virginicum wild. They reported that reaction of nine isolates of A. candida differed from each other on 26 differential hosts revealing thereby, that the monotypic pathogen A. candida on crucifers existed in the form of different biological races designated as new biological races or forms 1 to 9. The concept of races in A. candida. as proposed by Pound and Williams (1963) was based on species relationships. Studies have, however, clearly demonstrated that cultivars of Brassica crops must be included in a set of host differentials to distinguish isolates of the pathogen within a present accepted race (Burdyukova, 1980; Pidskalny and Rimmer, 1985). There is an urgent need to standardize host differentials keeping in mind the homogenity and purity of species and varieties. Petrie (1988) using North American race 2 and 7 from B. juncea and B. rapa, respectively, have screened accessions of several Brassica species including B. rapa var. Yellow Sarson, B. rapa var. Brown Sarson, B. rapa var. Toria and B. juncea from India, both yellow and brown sarson, were equally highly susceptible to both races, toria only to race 7, and B. juncea only to race 2. A detailed study is needed to determine whether the races of A. candida attacking B. juncca and several B. rapa crop in India are similar to race 2 and 7 from Canada and the USA. Kolte et al. (1991) reported that the WR isolate obtained from B. rapa appeared to be distinct in pathogenicity from the one obtained from B. juncea under Indian conditions. Petrie (1994) in Saskatchewan and Alberta, Canada discovered new races 7v in 1988 and race 2v in 1989. Verma et al (1999) reported two new races of A. candida in India viz., race 12 from B. juncea and race 13 from B. rapa var. Toria using 14 (including 6 standard) crucifer host differentials. Mathur et al. (1995b) and Rimmer et al. (2000) collected isolates of A. candida from different geographic locations in Western Canada and tested virulence on a number of cultivars and accessions of Brassica species. Most isolates were identified as race 7, which could be subdivided into 7a and 7v on the basis of their virulence on B. rapa cv. Reward. Isolates 28-7 and 29-1 were avirulent to all the differentials except the rapid cycling B. rapa CrGCI-I8. Tower isolates, 11-6 and 41-4, which could infect cultivars of both B. rapa and B. juncea, appeared to be hybrids between race 2 and race 7. Wu et al. (1995) studied genetic variation among isolates of A. candida using randomly amplified polymorphic DNA (RAPD) with five selected random primers fingerprint patters generated for each isolates. Most polymorphism was found between different races than among isolates within a single race. Most Canadian field isolates were grouped as race 7 and could be further subdivided into two groups (7a and 7v). Classification of A. candida isolates based on the results from the RAPD analysis was identical to the virulence classification on 10 Brassica differentials. Four distinct and new pathotypes of A. candida viz, ACI4 from RL 1359, AC 15 and AC 16 from Kranti, and AC 17 from RH 30 cultivars of B. juncea have been identified on the basis of their differential interactions on 11 host differentials by Gupta and Saharan (2002). Jat (1999) identified 20 distinct pathotypes of A. candida, 17 from B. juncea (AC 18 to AC 34), 2 from B. rapa var. Brown Sarson (AC 35 to AC 36), and one from B. nigra (AC 37). From Western Australia, Kaur et al. (2008) identified pathotype AC 2A from B. juncea and pathotype AC 2v from’ Raphanus raphanistrum. The pathogenic variability recorded in A. candida in the form of races are: 2 from Australia, 20 from Britain, 4 from Canada, 2 from Germany, 49 from India, 8 from Japan, 18 from Rumania and 7 from USA. However, nomenclature of A. candida races came into practice after the use of host differentials to distinguish races by Pound and Williams (1963). Global virulence of A. candida based on primary host is documented in Table 2. In A. candida, the sexual reproduction in the form of oospores is very common especially on B. juncea. Therefore, numerous races are expected to exist. In addition to this, other mechanism of variability including recombination, mutation and heterokaryosis are also in operation in the nature. To get the true picture of A. candida races and virulence spectrum, there is Journal of Oilseed Brassica, 5 (special) : Jan 2014 21 Table 1: Global virulence of A. candida pathotypes (Saharan, 2010) Pathotype designate Country International primary host Reference AC1 North America Raphanus sativus Pound and Williams, 1963 AC2 North America Brassica juncea Pound and Williams, 1963 AC2V North America B. napus Petrie, 1994 AC3 North America Armoracia rusticana Pound and Williams, 1963 AC4 North America Capsella bursa-pastoris Pound and Williams, 1963 AC5 North America Sisymbrium officinale Pound and Williams, 1963 AC6 North America Rorippa islandica Pound and Williams, 1963 AC7 North America B. rapa Verma et al., 1975 AC7V North America B. rapa cv. Reward Petrie, 1994 AC8 North America B. nigra Delwiche and Williams, 1977 AC9 North America B. oleracea Williams, 1985 AC10 North America Sinapis alba Williams, 1985 AC11 North America B. carinata Williams, 1985 AC12 India B. juncea Verma et al., 1999 AC13 India B. rapa var. Toria Verma et al., 1999 AC1 to 9 India Brassica species Singh and Bhardwaj, 1984 AC1to 5 India Brassica species Lakra and Saharan, 1988c AC14 India B. juncea cv RL 1359 Gupta and Saharan, 2002 AC15 India B. juncea cv Kranti Gupta and Saharan, 2002 AC16 India B. juncea cv Kranti Gupta and Saharan, 2002 AC17 India B. juncea cv RH 30 Gupta and Saharan, 2002 AC18 to 34 India B. juncea cv RH 30; EC 182925; DVS 7-3-1 Jat, 1999 AC35 and AC36 India B. rapa var. Brown Sarson Jat, 1999 AC37 India B. nigra Jat, 1999 AC2A Western Australia B. juncea cv Vulcan; Commercial Brown Kaur et al., 2008 AC2V Western Australia Raphanus raphanistrum Kaur et al., 2008 22 Journal of Oilseed Brassica, 5 (special) : Jan 2014 an urgent need to standardize host differentials for each crucifer species in the form of isogenic lines at international level. Standard nomenclature of the races viz, Acjun 1, 2,- for B. juncea isolates, AC rap 1, 2- for B. rapa isolates, AC nig 1,2 - for B. nigra isolates and ACol 1,2, - for B. oleracea isolates, and so on, appears to be a very useful beginning. 13.1 Virulence spectrum of Albugo candida As per the gene-for-gene hypothesis, interaction of Albugo-crucifers for compatibility and incomtability phenotype determines number of virulence genes in the pathotype and resistance genes in the host genotype. It has been observed that pathotypes of A. candida from B. juncea have wide range of virulence genes. Pathotypes like AC 23, AC 24, AC 17 infects only one, two and three differential hosts respectively, indicating a limited virulence potential. However, pathotypes of wider virulence viz., AC 29, AC 27, AC 30, AC 18 and AC 21 infected 21, 18, 16, 12 and 10 host differentials, respectively (Jat, 1999; Gupta and Saharan, 2002). Availability of virulence variability in pathotypes from B. juncea has suggested the possibility of identification of more number of resistant genes in the genotypes including identification of loci and alleles. In the absence of isogenic lines, it is not clear weather the races with wider virulence attack the same genes in the differentials, or genes for susceptibility are different or situated on different loci or tightly linked. 14. Host resistance The transfer of resistance from different sources in Brassica crops is possible and is being done through conventional as well as modern technologies all over the world (Saharan et al., 2005). 15. Genetics of host-parasite interactions Studies on the genetics of host-parasite interactions in WR disease have been concentrated largely on the level of host genotypes without considering pathogens’ races. Although, All India Co-ordinated Research Project on Rapeseed-Mustard (AICRPRM) have identified several genotypes with stable resistance, but very few have been utilised for developing WR resistant cultivar (Table 2). Thus, understanding of Brassica genotypes by A. candida interactions is of vital importance in identifing resistant genotypes for specific adaptability. GSL-1, EC 414299 and EC 399299 showed additive gene for horizontal resistance to WR which can prove good donors in further genetic improvement programmes. Varuna, JMM 07-2, JMM 027-1 and JYM 10 had non-additive gene action for pathogenicity to WR. PBC 9221, GSL 1, EC 414299 and EC 399299 were very similar in genetic make-up for disease resistance while Varuna showed maximum divergence in genetic constitution from these strains (AICRPRM, 2009). Even within the confines of race cultivar specificity, the studies have been one-sided in that no genetic information has been generated on Albugo, the causal organism. Interest in such studies was stimulated by Hougas et al. (1952), who investigated on the genetic control of resistance in WR of horse radish. The exhaustive work of Pound and Williams (1963) clearly demonstrated that resistance to white rust was controlled by a single dominant gene in radish cv. China Rose Winter (CRW) and Round Black Spanish (RES). Histological studies revealed that resistance in CRW was manifested as a hypersensitive reaction, which might be modified to a sporulating tolerant reaction by environmentallycontrolled minor genes. Humaydan and Williams (1976) while studying the inheritance of resistance in radish to A. candida race 1, changed the gene designation R into the more descriptive symbol AC-l derived from the initials and race number of A. candida. The resistance to A. candida race 1 in Raphanus sativus cv. Caudatus was controlled by a single dominant gene, A C-l. The resistance gene AC-I and the gene Pi, controlling pink pigmentation was found to be linked with a recombination value of 3.20 per cent. Bonnet (1981) found that WR resistance in radish variety Rubiso-2 was also controlled by one dominant gene. Among Brassica species monogenic dominant resistance to A candida race 2 has been found in B. nigra. B. rapa, B. carinata and B. juncea (Delwiche and Williams, 1974; Ebrahimi et al., 1976; Thukral and Singh, 1986; Tiwari et al., Journal of Oilseed Brassica, 5 (special) : Jan 2014 1988). A single dominant gene, AC-2, controlling resistance to A. candida race 2 in B. nigra was identified by Delwiche and Williams (1981). In a study to select quantitatively inherited resistance to A. candida race 2 in B. rapa, CGS-l, Edwards and Williams (1982) found that variability in reaction to A. candida race 2 among susceptible B. rapa strain PHW-Aaa-l was due to quantitative genetic regulation and suggested that rapid progress in resistance breeding could be made via mass selection when starting with a susceptible base population. Canadian cultivars of B. napus were resistant to WR, but many cultivars of this species grown in China were susceptible (Fan et al., 1983). The inheritance of WR resistance in B. napus cv. Regent was conditioned by independent dominant genes at three loci, designated as AC-7-1, AC-7-2 and AC-7-3. Resistance was conferred by dominance at anyone of these loci, while plants with recessive alleles at all loci were susceptible (Fan et al., 1983). Verma and Bhowmik (1989) were in part agreement with those of Fan et al. (1983) who suggested that resistance of BN-Sel (B. napus) to the B. juncea pathotype of A. candida found in India was conditioned by dominant duplicate genes. The host-pathogen-interaction-genetics studies indicates, that resistance in host is governed by one, two or more than two dominant genes (AC-7-1, AC-7-2, AC-7-3), additive genes with epistatic effects, and single recessive gene (WPr) alongwith a single gene (WRR4) confirring broad spectrum resistance to races, AC-2, 4, 7 and 9 (Pound and Williams, 1963; Fan et al., 1983; Liu et al., 1996; Saharan and Krishnia, 2001; Bansal et al., 2005; Borhan et al., 2008). The inheritance of virulence in Albugo- Brassica system suggested that a single dominant gene controls avirulence in race AC-2 to B. rapa cv. Torch (Adhikari et al., 2003). Systemic resistance in B. juncea to A. candida can be induced by pre-or co-inoculation with an incompatible isolates of A. candida (Singh et al., 1999). Resistant genes have been mapped and identified on the chromosomes of B. juncea viz., ACr (Cheung et al., 1998), AC-21 (Prabhu et al., 1998), AC-2 (Varshney et al., 2004), ACB1-A4.1, ACB1- 23 a5.1 (Massand et al., 2010), B. rapa viz., ACA1 (Kole et al., 1996), B. napus viz., ACA1 (Ferreira et al., 1994), AC 2V1 (Somers et al., 2002) and A. thaliana viz. RAC-1, RAC-2, RAC-3 and RAC-4 (Borhan et al., 2001; 2008) effective against one or more than one race of A. candida. In a study of inheritance of resistance to A. candida race 2 in mustard, Tiwari et al. (1988) found that resistance was dominant, monogenic, controlled by nuclear genes, and was easily transferred to adapted susceptible genotypes via back crossing. In a study evaluating performance of 15 advanced generation (F6) progenies of two interspecific crosses of B. juncea and B. carinata against A. candida, Singh et al. (1988) showed significant differences among the hybrid progenies which all gave a resistant reaction. A later study on five interspecific crosses between B. juncea and B. carinata revealed that the dominant gene which conferred resistance to WR was located in C genome of B. oleracea a progenitor of B. carinata (Singh and Singh, 1988). Williams and Hill (1986), and Edwards and Williams (1987) have opened unusual potential for resolving many problems relating to host-parasite interactions and breeding for disease resistance through development of rapid cycling Brassica populations. Their preliminary studies demonstrated considerable isozyme variations among individuals in a population which when inoculated with several pathogens, showed a wide range of plant to plant variation in the levels of resistance and susceptibility. This will assist plant breeders in developing cultivars with genetic resistance to plant diseases. Gene pools of both major and minor genes for resistance to various crucifer pathogens have been constructed (Edwards and Williams 1987; Hill et al., 1988; Williams and Hill 1986) which will be of immense value to plant breeders seeking sources of resistance. Thukral and Singh (1986) studied the inheritance of WR resistance in two crosses involving resistant (R) and susceptible (S) types of B. juncea namely EC 12749 x Prakash and EC 12749 x Varuna under normal and late-sown cohditions and found that analysis of six generations revealed the importance of additive, domi- 24 Journal of Oilseed Brassica, 5 (special) : Jan 2014 nant and epistatic effects. Reciprocal recurrent selection was also advocated for exploiting the additive and non-additive gene effects for resistance to WR. Singh and Singh (1987) reported that when A. candida resistant Ethiopian mustard (B. carinata) was crossed with B. juncea, the interspecific hybrids showed tolerance to A. candida. In the study on the inheritance to A. candida race 7 in B. napus, Liu et al. (1987) found that a digenic model with dominant resistance is confirred by RJ and R2 gene. Presence of a dominant allele at either of the two loci will confer resistance to a plant, whereas homozygous recessive at both loci will result in a susceptible phenotype expression. Liu and Rimmer (1992) studied the inheritance of resistance to an Ethiopian isolate of A. candida collected from B. carinata using two B. napus lines and suggested that resistance to the B. carinata isolate was conditioned by a single dominant resistant gene. Pal et al. (1991) evaluated the genetic component of variation for WR resistance through a 12 x 12 diallel crosses involving resistant and susceptible parents of Indian and exotic origin mustard under four sets of environmental conditions viz, normalsown in natural conditions, normal-sown in artificially-created-epiphytotic conditions, late-sown in natural conditions and late-sown in artificiallyepiphytotic conditions. Based on these results, they suggested that in all four sets of environments, both additive and non-additive components of variation were significant but an over dominance under the late-sown environment. Gadewadikar et al (1993) in their study suggested that resistance to A. candida was governed by a single dominant nuclear gene pair which could easily be transferred via back crossing. Paladhi et al. (1993) also concluded that the resistance to A. candida in an Indian mustard genotype PI-15 was controlled by a single dominant gene. Bains (1993) reported that resistance in the leaves differed from that of resistance in the young flowers; in the leaves it was due to the CC genome transferred from Indian mustard. Rao and Raut (1994) observed that the susceptibility of B. juncea cv. Varuna to the local Delhi pathotype of A. candida was conditioned by two genes, with dominant and recessive gene interaction. Interspecific crosses between B. juncea and B. napus suggested that resistance in WW 1507 and ISN 114 to A. candida was controlled by a single dominant gene (Jat, 1999). In their study of three interspecific crosses between B. juncea and B. napus, Subudhi and Raut (1994) revealed digenic control with epistatic interaction for WR resistance trait and a close association of parental species and different grades of leaf waxiness. Sachan et al. (1995) in their study using diallel fashion crosses between two WR resistant Canadian B. juncea cvs. Domo and Cutlass, and two susceptible B. juncea Indian cvs. Kranti and Varuna, reported that F1 hybrids, except susceptible x susceptible, were resistant; segregation pattern for resistance in F2 and test crosses was under the control of a single dominant gene in Domo and Cutlass, and that a recessive gene for susceptibility was present in Kranti and Varuna. Liu et al. (1996) in Canada developed monogenic lines for resistance to A. candida from a Canadian B. napus cultivar, and suggested that these monogenic lines could be used to study the mechanism of resistance response conditioned by the individual genes. These lines also facilitate molecular mapping of the loci in B. napus for resistance to A. candida race 7. In an inter-varietal cross between a susceptible Indian cv. Pusa Bold and a resistant genotype DIRA 313. Mani et al. (1996) showed a significant additive x additive interaction for the a) final intensity of WR on plant (FIP), b) final intensity of WR on leaf (FIL), and c) area under disease progress curve (AUDPC) along with the association of complimentary epistatic interactions indicating close association between the nature of inheritance for AUDPC on one-hand, and FIP and FIL on the other. This was also substantiated by a significant correlation between FIP and FIL, and AUDPC suggesting ease in selection for lower AUDPC (slow rusting) through FIP or FIL. Sridhar and Raut (1998) reported a monogenic inheritance showing complete dominance in four crosses and lack of dominance in seven crosses attempted between B. juncea and resistance sources derived Journal of Oilseed Brassica, 5 (special) : Jan 2014 25 from different species. According to Jat (1999), the resistance was dominant in all the crosses except susceptible x susceptible where it was recessive. Under controlled conditions, inoculation with three different races of A. candida on F2 population of crosses from R x R revealed that the resistant genes may be located on the same locus or on different loci. In both intra-specific and interspecific crosses between B. juncea X B. carinata, Saharan and Krishnia (2001) showed that resistance was dominant in all the crosses. They confirmed that resistance to A. candida was governed by one dominant gene or two genes with either as dominant, recessive or epistatic interaction or complete dominance at both gene pairs. Partial resistance in B. napus to A. candida was controlled by a single recessive gene designated as wpr with a variable expression (Bansal et al., 2005). Dominant alleles at three unlinked loci (ACh AC7z, and AC73) conferred resistance in B. napus cv. Regent to race AC 7 of A. candida (Fan et al., 1983; Liu et al., 1996). Two loci also controlled resistance in B. napus to A. candida race AC2 collected from B. juncea (Verma and Bhowmik, 1989). The Chinese B. napus accession 2282-9, susceptible to AC7 has one locus controlling resistance to an isolate of A. candida collected from B. carinata (Liu and Rimmer, 1992). These studies Indicated that only one allele for resistance was sufficient to condition an incompatible reaction in this pathosystem (Ferreira et al., 1995). In addition, a single locus controlling resistance to AC2 in B. napus and B. rapa was mapped using restriction fragment length polymorphism (RFLP) marker (Ferreira et al., 1995). A dominant allele at a single locus or two tightly linked loci were reported to confer resistance to both races AC 2 and AC 7 of A. candida (Kole et al., 2002). According to Borhan et al. (2008), a dominant WR resistant gene, WRR 4, encodes a TIR-NB-LRR protein that confers broad-spectrum resistance in A. thaliana to four races (AC2, AC4, AC7 and AC9) of A. candida. powdery mass which can readily be dispersed by wind or rain drops to cause secondary infection. In rapeseed, WR pustules become visible in 5-6 days after inoculation (Liu et al., 1989), while in cabbage symptoms appear in 8 days after inoculation (Coffey, 1975). Slow-rusting requires longer incubation and latent periods. In B. juncea cvs. Rajat and RC 781, incubation and latent periods of 11/14, and 11/15 have been observed; similarly, in B. rapa cvs. Candle, Tobin and Span, longer incubation and latent periods of 11/115, 15/1l8, and 1l/ 18 days, respectively have been reported (Lakra and Saharan 1988d; Jat, 1999; Gupta and Saharan, 2002). There is a need to identify genotypes with slow-rusting attributes to curb the epidemic development of WR in the field. Partial resistance to A. candida in crucifer genotypes can be identified through lower infection frequency, lower spore production, and longer incubation and latent periods. 15.1 Slow white rusting in crucifers An oospore germination technique was used to study the effectiveness of 27 protectant fungicides in inhibiting oospore germination at various stages (Verma and Petrie, 1979). Among the chemicals tested, the three mercurial fungicides, mersil, PMA- Rate of infection or disease spread is influenced by incubation and latent periods of A. candida in its compatible host. In WR, the sporangia become visible after the host epidermis is ruptured as a white 16. Chemical control 16.1 Efficacy of fungicides on germination of A. candida oospores in vitro Albugo candida oospores occur as common contaminant in Brassica seed samples (Petrie, 1975). Inoculum levels on seeds may be considerably higher than actually required for initiation of infection considering that on germination a single oospore releases 40-60 zoospores (Verma and Petrie, 1975). Germination of oospores following a period of washing in water, infection of Brassica cotyledons by zoospores from germinating oospores, and field experiments showing more foliar and staghead infection in oospore-treated plots than in the controls, support the view that oospores contaminated seeds constitute a primary inoculum for infection of Brassica species (Verma et al.,1975). Thus treatment even by a protectant fungicide can be important in controlling WR infections either by inhibiting oospore germination or by killing the zoospores on emergence. 26 Journal of Oilseed Brassica, 5 (special) : Jan 2014 10 and panogen, were the best inhibitors of oospore germination. The total inhibition with any of these fungicides at a concentration of 500 ppm active ingredient (a.i.) was about 75 %. Among the nonmercurial compounds, mancozeb and ethazol were the most effective giving total inhibition of about 60%. The inhibition provided by bromosan and pyroxychlor was about 50 %. Since none of the fungicides tested in this study was 100 % effective, the search for a completely effective, preferably systemic, fungicide needs to be continued. 16.2 Efficacy of protectant fungicides in controlling both the foliar and staghead phase of WR disease Using protectant fungicides, several researchers around the globe have reported varied degree of control of A. candida-induced foliar infections in various cruciferous hosts (Verma and Petrie, 1979; Sharma and Sohi, 1982; Sharma and Kolte, 1985; Sharma, 1983; Singh et al., 2002; Dainello et al., 1986; 1990; Chambers et al., 1974, Singh and Singh, 1990; Meena and Jain, 2002; Pandya et al., 2000; Saharan et al., 1990). In a detailed growth chamber study, Verma and Petrie (1979) reported that of the nine protectant fungicides tested, application of either chlorothalonil or mancozeb, at 250 or 500 ppm, respectively, 6 h before inoculation and then a week later, controlled the disease effectively. In view of their mainly protectant action, failure to control WR by either fungicide applied 24 h and 7 days after inoculation was not surprising, as establishment of A. candida infection on rapeseed cotyledons, and perhaps leaves, would normally be completed within 24 h of inoculation (Verma et. al., 1975). the foliar and staghead phase of WR disease Among the systemic chemicals, metalaxyl is probably the best fungicide currently available for WR control. Metalaxyl was active against A. candida race 7 in B. rapa cv. Torch (Stone et. al., 1987a, b). Treating the seed with metalaxyl at 5.0 g a.i. /kg controlled foliar infection in the growth chamber up to the sixth leaf stage, 22 days after planting. When sprayed on the plants up to 4 days after inoculation, metalaxyl reduced foliar infection by 95 %. Foliar infection was also controlled when applied as a soil drench, but phyto-toxicity was evident. Foliar spray application at 2.0 kg a.i. /ha or higher reduced foliar infections in three years of field studies. Foliar applications also reduced staghead infections when applied at growth stages 3.2 or 4.1. Growth chamber and field studies (Stone et. al., 1987a) showed that metalaxyl possesses both protective and eradicative activity against A. candida. Control of disease in tissues remote from the site of application indicated that the fungicide moves systemically in rape plants. Disease control was obtained on the foliage, either by seed treatment or soil drenching, and disease eradication was successful when the fungicide was sprayed within 4days of inoculation, a further evidence of systemicity (Stone et. al., 1987b). The best cost benefit ratio was obtained by Mehta et al., (1996) when seed treatment with Apron SD-35 (2 g a.i. / kg) was followed by three sprays of mancozeb (0.2 %) at 40, 60 and 80 days after seeding. However, best disease control was obtained when three sprays of Ridomil MZ-72 (0.25 %) were given at 40, 60 and 80-days after seeding. Two foliar spraying of chlorothalonil (Bravo) in June under Canadian conditions when the plants were 3-4 weeks old significantly reduced both foliar and staghead infections in the field (Verma and Petrie, 1979). However, in view of the growth room studies on successful initiation of stagheads (Verma and Petrie, 1980), a third application at the time of flowering is also advised. Multiple applications, however, may not be economically feasible under commercial rapeseed production. Seed treatment results were promising but in field situation it provided adequate protection only in the early stages of plant growth. The decline in the activity of metalaxyl with increasing age of plants in seed treatment experiments may have been the result of fungicide dilution as the volume of plant tissue increased. Accordingly, infection of flower buds by wind-borne zoosporangia was not controlled by seed dressing. 16.3 Efficacy of metalaxyl in controlling both In the growth chamber, metalaxyl was active as a foliar eradicant for up to 4 days, but when applied 5 Journal of Oilseed Brassica, 5 (special) : Jan 2014 or 6 days after inoculation, the fungicide did not prevent sporulation (Stone et. al., 1987a). It would appear, therefore, that after 4 days the fungus had reached a stage of development when fungicide treatment could not completely arrest growth, although pustule size and development were restricted with these late applications. Sharma and Sohi, 1982; Sharma and Kolte, 1985; Sharma, 1983; Srivastava and Verma, 1989; Singh et al., 2002; Dainello et al., 1986; Singh and Singh, 1990; Meena and Jain, 2002, Mehta et al., 1996) have also been reported globally. 17. Suggestions for future research i. Results of studies by Verma and Petrie (1979, 1980) and Stone et al. (1987a) suggest that A. candida does not require early infections to develop systemically but can produce stagheads from infections of young flower buds by zoospores arising from wind-borne zoosporangia after plant growth stage 2.6. Successful disease control with metalaxyl, therefore, requires that a sufficient quantity of the fungicide be available well into the growing season. Seed dressings only provide protection for a limited period of time, and if conditions favour disease development throughout the season, staghead development will not be controlled. By providing early disease control, however, seed treatment could reduce the secondary inoculum potential in the crop, and thereby limit initiation of stagheads from newly infected flower buds. Bioassay and gas chromatographic analyses of plant tissue extract confirmed the presence of metalaxyl in tissue remote from the site of the treatment (Stone et. al., 1987b). Both bioassay and chemical analyses of plants grown in metalaxyl-drenched soil showed that the fungicide was readily taken up by plants from the soil solution, that the greatest accumulation was in the lower leaves, and that metalaxyl was found in decreasing amount in leaves furthrest from the roots and in only small concentrations in the stem and inflorescence. These results indicate that root absorption is an efficient means of metalaxyl uptake because when applied to a single leaf it was not detected in the leaves below or above the treated leaf; thus, it is concluded that negligible symplastic translocation occurs. Different levels of control of WR using metalaxyl treated seeds (Verma and Petire, 1979; Rod, 1985; Sokhi et al., 1997; Pathak and Godika, 2005), soil drenching (Stone et al., 1987a; Dainello et al., 1990) and foliar application (Verma and Petire, 1979; 27 Information regarding production of oospores inside the seeds, and their possible importance both in the survival and initiation of primary infection are lacking. ii. Role of simple or branched germ tubes from germinating oospores need to be studied. iii. Single zoospore cultures from germinating sporangia and oospores must be prepared and their pathogenicity compared. iv. After screening lines for resistance against foliar infections, some select advanced lines must also be screened for production of stagheads using flower-bud inoculation technique. v. Based on host specificity, mycologists may consider classifying A. candida complex into different species. vi. Host differentials in each crucifer species in the form of isogenic lines must be standardized internationally. vii. Nomenclature of the A. candida races should be standardized internationally viz. AC jun I, 2for B. juncea, AC rap 1, 2, - for B. rapa, AC nig I, 2, - for B. nigra, A C ol 1,2, etc., for B. oleracea. viii. Identification of sources of resistance should be based on broad spectrum effectiveness of a genotype against specific races, and inheritance of resistance should be studied alongwith the virulence spectrum of A. candida isolates. ix. Efforts should be made to identify resistant loci in the genotypes along with alleles for resistance in each locus. x. Genotypes exhibiting attributes of slow white-rusting, disease tolerance, and partial resistance may be categorized. 28 Journal of Oilseed Brassica, 5 (special) : Jan 2014 xi. 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