5.1 Introduction

Specificity in the downy mildew fungus on crucifers is very complex since it occurs on a wide range of wild hosts as well as agricultural and horticultural species. However, there has been little sustained effort to introduce resistance to the disease, and hence there has been less selection pressure exerted on the pathogen population than is the case with many other obligate parasites . Further impetus has been added by the exponential growth in research on the wild crucifer Arabidopsis thaliana, as a host for Hyaloperonospora parasitica and serving as a model system for genetic and molecular analysis (Uknes et al. 1992). Discontinuities in the host range of isolates from different host genera and species suggest that the fungus may exist as a series of pathotypes adapted to each host of origin, although some cross-infections may occur. There is also growing evidence that within host species, specificity may be determined by genotype-specific interactions consistent with a gene for gene recognition system (Lucas et al. 1988, 1994; Nashaat and Awasthi 1995). Specificity might therefore be expressed at several levels including family, genus, species, and cultivar or accession . In view of the close cytogenetic relationship between the major Brassica species , coupled with the strongly outbreeding nature of several of these, some overlap in the host range of species-adapted isolates is perhaps predictable.

At the generic level, pathogenic specialization has been observed by several workers all around the world. Gardner (1920)) and Kobel (1921) suggested that H. parasitica is highly specialized and seldom occurs in the same biological form on more than one crucifer. An isolate of H. parasitica obtained from turnip is able to infect seedlings of turnip but not rutabaga or radish (Gardner 1920). In Holland disease on cabbages is classified in two groups, both representing distinct biological forms of the fungus. The first is characterized by short, ellipsoid conidia and the second by larger, elongated conidia with protuberant apices. The average dimensions of the later group are 32.51 × 25.66 μm and that of the former 26.67 × 23.13 μm, the corresponding ratios of length to breadth being 1.26 and 1.11, respectively (Thung 1926a). However, Gaumann (1926) subdivided H. parasitica (= H. brassicae) into three biological strains , namely, 1. f. sp. brassicae, the chief hosts of which are B. oleracea, B. napus, B . rapa, B. nigra , B. juncea , B. tournefortii , and B. fruticulosa, but can also cause some infection on Sinapis arvensis, S. alba, Raphanus raphanistrum , R. sativus, and Eruca sativa ; 2. f. sp. sinapidis, the principal hosts of which are S. arvensis and S. alba, but are also able to produce sub-infections on all the above-mentioned species of Brassica (except B. rapa and B. juncea) and Raphanus, with occasional conidiophore formation on B. oleracea; and 3. f. sp. raphani, the chief hosts of which are R. raphanistrum and R. sativus, but can also produce sub-infections on all the above-mentioned species of Brassica (except B. fruticulosa) , as well as on S. arvensis and S. alba, with occasional conidiophore formation on B. oleracea and B. napus. The downy mildew on radish does not attack cabbage (B. oleracea var. bullata and capitata) and is slightly pathogenic on Chinese cabbage (B. pekinensis , B. chinensis), rape ( B. campestris ), and mustard (B. juncea) (Hiura and Kanegae 1934). Conversely, the form derived from B. pekinensis does not infect radish but is allied to one on B. chinensis and rape. The forms derived from B. pekinensis, B. chinensis, and rape are mutually pathogenic on one another (Hiura and Kanegae 1934).

5.2 Pathogenic Variability

The identification of pathogenic variability seems to be initiated in 1944 with the observation of Wang (1944) who classified the reaction of the hosts into four categories: 1. susceptible with normal symptoms; 2. resistant , showing large necrotic spots; 3. para-immune , showing slightly visible necrotic dots; and 4. immune, with no visible symptoms. Three pathotypes of H. parasitica were differentiated: H. parasitica Brassicae on Brassica, H. parasitica Raphani on Raphanus, and H. parasitica Capsellae on Capsella. The three pathotypes were not mutually compatible with each other’s host. Six forms of H. parasitica Brassicae were differentiated by their reaction to B. chinensis, B. oleracea, B. juncea , and B. napobrassica . Wang (1944) prepared a dichotomous key to physiological forms from China as follows:

A.:

Capsella bursa -pastoris, immune

B.:

Raphanus sativus , immune or para-immune variety Brassicae

C.:

B. oleracea, resistant or para-immune

D.:

B. chinensis, susceptible

E. :

B. juncea (Meitan Dav Yu Tsai), susceptible Ph.fm. 1

EE.:

B. juncea (Meitan Dav Yu Tsai,) resistant

F.:

B. napobrassica, immune Ph. fm. 2

FF.:

B. napobrassica , resistant Ph. fm. 3

DD.:

B. chinensis, resistant

E.:

B. juncea (Dav Ching Tsai), susceptible Ph. fm. 4

EE.:

B. juncea (Dav Ching Tsai), resistant Ph. fm. 5

CC.:

B. oleracea, susceptible Ph. fm. 6

BB.:

R. sativus, susceptible variety Raphani

AA.:

C. bursa-pastoris, susceptible variety capsellae

Felton and Walker (1946) and Natti (1958) differentiated the races of H. parasitica found on R. sativus and B. oleracea on the basis of their host specificity. Morris and Knox-Davies (1980) also indicated distinct races of H. parasitica on B. oleracea and R. raphanistrum based on host specificity. H. parasitica f. brassicae on cabbage, f. rapae on turnip, f. rapifera on B. rapa, f. rapifera , f. napi on rape, f. raphani on radish , and f. sinapsidis on Sinapis alba have been distinguished as special forms of H. parasitica from Leningrad though all are similar morphologically (Dzhanuzakov 1963).

Three vars. of H. parasitica have been differentiated in 35 samples of downy mildew from B. pekinensis and other crucifers, namely, f. sp. brassicae on Brassica, f. sp. raphani on Raphanus, and f. sp. capsellae on Capsella (Chang et al. 1964). Hyaloperonospora parasitica f. sp. brassicae exists in at least three different subforms (pekinensis, oleracea, and juncea). Isolates from B. pekinensis, B. chinensis , and turnip were classified in the same group and can attack all three hosts but did not infect Capsella bursa-pastoris , radish , cabbage, Chinese mustard, and B. juncea var. multiceps. B. juncea var. megarrhiza expressed various reactions to these isolates. Isolates from Chinese mustard, B. juncea var. megarrhiza and B. juncea var. multiceps , were limited to these hosts, except that those from Chinese mustard did not infect some vars. of B. pekinensis , B. chinensis, and turnip . Radish isolates are of two types, one infects only radish, the other vars. of B. pekinensis, cabbage, and turnips as well as radish. Isolates from cabbage and C. bursa-pastoris are host specific. In Norway, cross-inoculation experiments with downy mildew from cabbage, turnip rape , and radish indicated the occurrence of different races on cabbage and radish (Semb 1969).

According to Natti et al. (1967), the predominant physiologic race of H. parasitica pathogenic to broccoli and other types of B. oleracea grown commercially in New York were race 1 and race 2. The later race was pathogenic to plants resistant to race 1. Dickinson and Greenhalgh (1977) observed a wide variation in the reaction of seedlings of different crucifers’ species to isolates of Hyaloperonospora derived from Brassica and Raphanus species (Table 2.3). Masheva et al. (1996a) found that the pathogenicity of two isolates of H. parasitica (one from Plovdiv and one from Goma Oriahovitsa, Bulgaria) towards ten hybrids of cabbage, broccoli, and cauliflower differed in pathogenicity and host specificity.

In India, H. parasitica isolates from different hosts vary in host range . Isolates from Brassica, Raphanus, Eruca, and Sisymbrium are not cross-infective (Bains and Jhooty 1983). Mehta and Saharan (1994) tested the host range of 9 isolates of H. parasitica collected from the leaves and stag heads of 6 host species on 17 host differentials (Tables 5.1 and 5.2). Isolates from Brassica oilseeds infected all species, except B. alba , whereas isolates from cauliflower leaves did not infect B. carinata , B. alba, B. nigra , B. chinensis , B. pekinensis , and B. napus (Table 5.2). There was no significant difference among the conidial size of the isolates collected from leaves and stag heads, but significant differences were observed among these groups (Table 5.3). The isolates were classified into two distinct pathotypes, one from cauliflower and the other from oilseeds Brassica. There was no significant difference between the isolates in percentages of spore germination (Table 5.4). Specific populations of H. parasitica differing in pathogenesis and host specificity were reported from Bulgaria (Masheva et al. 1996a, b). The populations formed at a lower temperature were more aggressive on cabbage heads.

Table 5.1 Host differentials of Hyaloperonospora parasitica (Mehta and Saharan 1994)
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Table 5.2 Response of seventeen Brassica species to nine isolates of Hyaloperonospora parasitica (Mehta and Saharan 1994)
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Table 5.3 Conidial size of Hyaloperonospora parasitica isolates derived from eleven Brassica species (Mehta and Saharan 1994)
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Table 5.4 Percent conidial germination of Hyaloperonospora parasitica isolates at 18°C (Mehta and Saharan 1994)
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In the UK, differential host resistance in relation to pathogenic variation of isolates derived from the same host species was identified in B. rapa (Moss et al. 1991; Silve et al. 1996), B. napus (Nashaat and Rawlinson 1994), B. juncea (Nashaat and Awasthi 1995), and B. oleracea (Silve et al. 1996). Isolates from different Brassica species found to be most virulent on their species of origin were nevertheless able to grow to less extent on other Brassica species (Sherriff and Lucas 1990). Nashaat and Awasthi (1995) identified five groups of B. juncea accessions with differential resistance to UK isolates Rl and P003 derived from oilseed rape (B. napus ssp. oleifera ) and Indian isolates IP01 and IP02 derived from mustard (B. juncea) (Table 5.5). All B. juncea accessions were resistant to isolates from B. napus , but at the same time, B. napus cv. Arian was resistant to isolates from B. juncea. Twenty-one differential responses to H. parasitica isolates from B. oleracea and two from B. rapa were identified. Of the seven isolates tested, four were from crops of cauliflower in France, two from oilseed rape in the UK, and one was from mustard in India. All Raphanus sativus accessions were resistant to all seven isolates (Silve et al. 1996).

Table 5.5 Sources of seed for accessions of Brassica juncea, arranged in five groups according to the response of their seedlings at the cotyledon stage to Hyaloperonospora parasitica and one accession of B. napus (Nashaat and Awasthi 1995)
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Differential resistance of rapid cycling and commercial genotypes of B. rapa were also reported from the UK (Moss et al. 1988). A range of differential host responses were characterized by four homologous isolates (Table 5.6). Pathogen isolates were also characterized in relation to their sexual compatibility type and response to phenylamide fungicides (Moss et al. 1988).

Table 5.6 Differential virulence of Hyaloperonospora parasitica isolates from B. campestris on six host lines (Moss et al. 1988, 1991)
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Eleven isolates of H. parasitica tested on rapid cycling populations of B. rapa (aa, CrGc-1-1), B. nigra (bb, CrGc-2-1), B. oleracea (cc, CrGc-3-1), B. juncea (aabb CrGc-4-1), B. napus (aacc CrGc-5-1), B. carinata (bbccCrGc-6-1), and R. sativus (rr CrGc-7-1) indicated specificity towards particular genotypes within each rapid cycling population (Hill et al. 1988). Variation in the response of different host lines to H. parasitica has also been detected within wild crucifer species such as shepherd’s purse ( Capsella bursa-pastoris ) and Arabidopsis thaliana (Lucas et al. 1994).

Moss et al. (1994) attempted to cross fungal isolates originating from different Brassica spp. by co-inoculating host lines previously identified as susceptible to these isolates . A proportion of oospore progeny recovered from these crosses appeared to be hybrids and had reduced virulence on both hosts of origin. The differential resistance to H. parasitica identified in Brassica spp. can be used for future studies of the genetics of the host-pathogen interaction and for breeding for disease resistance.

The pathogenicity of four isolates of H. parasitica obtained from Japanese radish, rape , broccoli, and shepherd’s purse (Capsella bursa-pastoris) was compared by observing infected cotyledons. These isolates eventually sporulated on host plant cotyledons (compatible combinations), but formed only small necrotic lesions on non-host cotyledons (incompatible combinations). Sheath encasement of haustoria and host cell death frequently occurred in the incompatible combinations. The C. bursa-pastoris isolate could only infect the host plant with haustorium formation in non-hosts inhibited mainly by papillae. Cytochemical observation showed that these papillae contained callose and polyphenol compounds but not lign in (Yoshida and Ohguchi 1998).

Silve et al. (1996) identified 21 differential responses to H. parasitica isolates from B. oleracea and 2 from B. rapa. All Raphanus sativus accessions were resistant to all seven isolates . Accessions for which seedling populations exhibited a heterogeneous reaction to some isolates were classified in a separate category. However, Mehta and Saharan (1994) classified 9 isolates of H. parasitica (collected from Brassica species ) into 2 distinct pathotypes, 1 from cauliflower, and the other from oil-yielding Brassica species on the basis of their reaction to a set of 17 host differentials .

Variation has been found among interaction phenotypes when a range of A. thaliana (At) and Brassica accessions are inoculated with different isolates of H. parasitica (Hp). In Brassica, genetic characterization of this variation was rudimentary, and only one allele at a single locus associated with isolate-specific resistance to H. parasitica in B. napus has been defined. In contrast, and considering only those phenotypes that can be discriminated readily among segregating individuals, alleles controlling genotype-specific variation for response to these pathogens have been identified at 21 loci (RPP and RAC) in the At genome. Most of these loci now have been mapped with varying degrees of resolution and occur in several linkage groups in four of the five At chromosomes (Leckie et al. 1996).

Eight B. oleracea lines were evaluated for their phenotypic reaction to the thirteen European isolates of H. brassicae (Table 5.7) by Coelho et al. (2012). The host lines exhibiting a similar phenotypic pattern to different isolates were clustered into six classes (A–F) according to their response to the isolates (Table 5.8). The control lines cv. Senna were highly susceptible with no visible host response to all of the pathogen isolates tested (designated class A) mean IP≥5.9, showing a consistent susceptible response (Tables 5.9 and 5.10). The two class F lines (PCA 20.14 and EBH 527) were resistant or moderately resistant to all thirteen isolates (mean IP≤3.0). The remaining five lines exhibited resistance to at least one, but not all of the isolates including a single line in class B (EBH508) which exhibited resistance to three isolates (Hp006, Hp 539, and Hp 704), and were highly susceptible to the remaining isolates: a class C line (EBH 502) which was resistant at least to some degree to seven isolates, a class D line (EBH 525) which was fully resistant to eleven isolates, and two class E lines (PC10 and PC11) which were resistant to all of the isolates except for high susceptibility to the single FP 06 isolates (from France).

Table 5.7 Brassica oleracea standard host differentials to classify pathotypes of Hyaloperonospora brassicae (H. parasitica) (Coelho et al. 2012)
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Table 5.8 Mean interaction phenotypes in seedlings following cotyledon inoculations for different combinations of eight Brassica oleracea lines and thirteen European isolates of Hyaloperonospora brassicae (H. parasitica) (Coelho et al. 2012)
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Table 5.9 Origin of the Hyaloperonospora brassicae (H. parasitica) isolates collected from field samples on different crop types of Brassica oleracea (Coelho et al. 2012)
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Table 5.10 Interaction-phenotype scores used to evaluate the response of Brassica oleracea cotyledons and relative amount of sporulation following inoculation with Hyaloperonospora brassicae (H. parasitica) (Coelho et al. 2012)
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Six different pathotypes of H. brassicae were identified by classifying the mean IP as either being compatible (mean IP ≥4.5), incompatible (mean IP <2.5), or intermediate (Table 5.8). Isolates showing the same virulence pattern on the differential set of host lines were grouped and considered to belong to the same pathotype. Pathotypes 1, 4, and 6 were represented by a single isolate, Hp006, Hp806, and FP06, respectively. Other pathotypes were represented by more than one isolate including two examples of pathotype 2 (Hp539 and Hp704), five of pathotype 3 (Hp 530, Hp533, Hp535, Hp717, and Hp710), and three of pathotype 5 (Hp541, Hp702, and Hp801). The three referred cases with multiple isolates included examples from different countries. For example, pathotype 3 includes isolates from Italy and two from diverse locations in Portugal (Odemira and Azores Island) and the UK (Coelho et al. 2012). Tham et al. (1994) used RAPD analysis to compare 16 isolates of H. parasitica from two different Brassica species , B. napus and B. oleracea. Two out of twenty random primers screened gave reproducible band patterns capable of discriminating between the different host-adopted isolates (Plate 5.1).

Plate 5.1
figure 1

Random amplified polymorphic DNA (RAPD) from 16 isolates of crucifer downy mildew ( Hyaloperonospora parasitica ), lanes 2–13 are isolates from oilseed rape Brassica napus, and lanes 14–17 are isolates from cauliflower B. oleracea (Tham et al. 1994)

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5.2.1 DNA Fingerprinting of H. parasitica

Different pathotypes can be distinguished on the basis of host specificity (Sherriff and Lucas 1990), but such tests are time-consuming and may not reveal the full extent of variations present. Other phenotypic markers such as sexual compatibility type (Sherriff and Lucas 1989a, b) or fungicide sensitivity (Crute et al. 1985) can be assessed, but they provide limited information for epidemiological studies or genetic analysis. There is need, therefore, for alternative molecular markers (Michelmore and Hulbert 1987) to further define variations in the pathogen.

Randomly amplified polymorphic DNAs (RAPDs) have been proposed as genetic markers that overcome many of the technical limitations of restriction fragment length polymorphism (RFLP) analysis (Williams et al. 1990; Welsh and McClelland 1990). RAPD scan can be used in the constructions of linkage maps (Williams et al. 1990; Reiter et al. 1992), in the identification of strains , and varieties by genomic fingerprinting (Welsh and McClelland 1990; Goodwin and Annis 1991; Hu and Quiros 1991; Schafer and Wostemeyer 1992; Kresovich et al. 1992; Klein-Lankhorst et al. 1991; Koller et al. 1993; Stiles et al. 1993) and following the cloning of amplified fragments may also serve as conventional RFLP probes. In the study carried out by Tham et al. (1994), the potential use of RAPDs as a source of genetic markers in H. parasitica was evaluated. Several isolates from different host species were compared to determine whether reproducible banding patterns correlated with host specificity. As H. parasitica can only be cultured on living plant tissues, particular attention was paid to possible artefacts arising through contamination of sample DNA from plant or other microbial sources.

Random amplified polymorphic DNA (RAPD) fingerprints were generated from a range of H. parasitica isolates from different Brassica species (Table 5.11). Reaction conditions, in particular DNA template, primer, and Mg2+ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus. Rehmany et al. (2000) compared downy mildew isolates from A. thaliana and B. oleracea using AFLP and ITS analysis dividing the isolates into five groups that correlated with taxonomic species and in most of the cases with host origin.

Table 5.11 Isolates used for RAPD analysis (Tham et al. 1994)
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5.2.2 Identification of Host Differentials and Nomenclature of Pathotypes

Physiologic specialization /pathogenic variability have long been recorded in H. parasitica infecting cruciferous plants all over the world (Gardner 1920; Kobel 1921; Thung 1926b; Gaumann 1926; Hiura and Kanegae 1934; Wang 1944). However, nomenclature and designation of H. parasitica pathotypes/races were initiated by Natti et al. (1967) based on differential interaction of isolates on cruciferous hosts, viz. broccoli and B. oleracea. Natti et al. (1967) identified and designated race 1 and race 2 of H. parasitica showing differential reactions on these two hosts. Later, several reports have come from different countries on the host specificity of H. parasitica isolates collected from numerous cruciferous hosts (Felton and Walker 1946; Natti 1958; Dzhanuzakov 1963; Chang et al. 1964; Semb 1969; Dickinson and Greenhalgh 1977; Morris and Knox-Davies 1980; Hill et al. 1988; Sherriff and Lucas 1990; Moss et al. 1991; Mehta and Saharan 1994; Lucas et al. 1994; Tham et al. 1994; Silve et al. 1996; Masheva et al. 1996b; Yoshida and Ohguchi 1998). The methodology and host species/varieties/lines used in these studies were not comparable and reproducible in the absence of non-maintenance of isolates and the use of international standard set of host differential . It is difficult to compare the results and determine whether distinct pathotypes are the same or different. Mehta and Saharan (1994) designated distinct pathotypes, one from cauliflower and other from oilseed Brassica on the basis of differential interaction of 9 isolates on a set of 17 host cruciferous host differential set (Table 5.12). Nashaat and Awasthi (1995) identified five groups of B. juncea (Table 5.5) accessions with differential resistance to UK isolates R1 and P003 derived from oilseed rape (B. napus ssp. oleifera) and Indian isolates IP01 and IP02 derived from mustard (B. juncea). Coelho et al. (2012) assembled set of genetically uniform lines of B. oleracea (doubled haploid or self-pollinated inbred lines) to use as host differential set for the characterization of pathotypic variation of H. parasitica (H. brassicae) within a European sample of the pathogen collected from various crop types of B. oleracea. Six pathotypes (HP1–HP6) were distinguished indicating a potential use of the host differentials for monitoring frequencies in H. parasitica populations (Table 5.8). A naming system for the isolates of H. arabidopsidis was introduced by Dangl et al. (1992), Holub et al. (1994), and Slusarenko and Schlaich (2003) on the basis of geographical location and ecotypes infected (AHCO, ASWA, CALA, EDCO, EMWA, etc.). For detailed description, see chapter 1, section 1.11.6. However, still there is a need to standardize a set of host differentials (isogenic lines) and a system of nomenclature and designation of pathotypes of H. parasitica at international level. It is essential to know whether pathotypes of H. parasitica reported so far from different countries are the same or different (Table 5.12). It will be very helpful to identify resistant genes and their combinations to breed resistant cvs. and their durability extent under different agroecological situations.

Table 5.12 Identification of pathotypes of Hyaloperonospora parasitica and H. arabidopsidis
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5.3 Heterothallism and Homothallism

Induction of the sexual process in several downy mildew species requires the presence of two strains of opposite mating type. Such heterothallic behaviour has been reported for H. parasitica (De Bruyn 1937; McMeekin 1960; Kluczewski and Lucas 1983; Sherriff and Lucas 1989b; Sequeira and Monteiro 1996). Homothallic forms of H. parasitica have also been observed (De Bruyn 1937; Sherriff and Lucas 1989b; Sequeira and Monteiro 1996). No relationship has been observed between geographic origin and mating types. Both heterothallic and homothallic isolates have been isolated from the same fields (Sequeira and Monteiro 1996). The two forms of sexual reproduction are very important for the maintenance and evolution of the fungal strains and epidemiology of the disease. Two mating type heterothallic isolates of B. oleracea and B. campestris designated as P1 and P2 have been identified. Isolates from oilseed rape B. napus have been found to be uniformly homothallic and remained self-fertile even after months of laboratory subculture (Sherriff and Lucas 1989b). In a cytogenetic study of heterothallic and homothallic isolates of H. parasitica at metaphase 1 of meiosis, a ring of four chromosomes is found (Sherriff and Lucas 1989a). This ring is interpreted as a reciprocal translocation complex between chromosomes carrying the mating-type alleles. In homothallic isolates a fifth chromosome is associated with the ring of four. The self-fertility of these isolates may therefore be due to the presence of a third mating-type allele on the fifth chromosome, a condition known as secondary homothallism . The determination of sexual compatibility type (SCT) of an unknown isolate can be achieved by mixing conidia in a 1:1 ratio with isolates of known SCT and inoculating to a common compatible host. In heterothallic isolates, oospores may form in combination with isolates of opposite SCT. Isoenzyme markers are particularly important in discriminating between self and true hybrid progeny (Moss et al. 1988).

5.4 Hybridization of Hyaloperonospora Isolates

Moss et al. (1994) produced viable oospore of H. parasitica under laboratory conditions and described recovered isolates (referred as sexual progeny) from these oospore populations. Oospores were produced when isolates of opposite sexual compatibility type, specialized to the same or different Brassica species , were grown together in seedling cotyledons of a host line capable of supporting growth of both isolates. Recovery of sexual progeny from oospore populations produced from two out of four pairing between isolates, is specialized in the same host species (homologous pairings) and proved to be relatively easy. On the basis of their characterization with respect to virulence, response to phenylamide fungicides, sexual compatibility type, and isoenzyme polymorphisms, there was evidence that the sexual progeny from these homologous pairing could be of hybrid origin. For the first time in a member of the Peronosporaceae, it provided possibility to recover and successfully characterize a new sexual progeny from pairing between isolates specialized to different host species (heterologous pairing). However, the majority of such isolates sporulated weakly and, as consequence, proved difficult to maintain and were lost. Nevertheless, it is concluded that some evidence for the hybrid nature of progeny from heterologous pairing was obtained.