Isolate-Dependent Inheritance of Resistance Against Pseudoperonospora cubensis in Cucumber

: Six wild accessions of Cucumis sativum were evaluated for resistance against each of the 23 isolates of the downy mildew oomycete Pseudoperonospora cubensis . The isolates originated from Israel, Europe, USA, and Asia. C. sativum PI 197088 (India) and PI 330628 (Pakistan) exhibited the highest level of resistance against multiple isolates of P. cubensis . Resistance was manifested as reduced lesion number, lesion size, sporangiophores and sporangia per lesion and enhanced encasement of haustoria with callose and intensive accumulation of lignin in lesions of both Plant Introductions (PIs) compared to the susceptible C. sativum SMR-18. In the ﬁeld, much smaller AUDPC (Area Under Disease Progress Curve) values were recorded in PI 197088 or PI 330628 as compared to SMR-18. Each PI was crossed with SMR-18 and o ﬀ spring progeny plants were exposed to inoculation with each of several isolates of P. cubensis in growth chambers and the ﬁeld during six growing seasons. F1 plants showed partial resistance. F2 plants showed multiple phenotypes ranging from highly susceptible (S) to highly resistant (R, no symptoms) including moderately resistant (MR) phenotypes. The segregation ratio between phenotypes in growth chambers ranged from 3:1 to 1:15, depending on the isolate used for inoculation, suggesting that the number of genes, dominant, partially dominant, or recessive are responsible for resistance. In the ﬁeld, the segregation ratio of 1:15, 1:14:1, or 1:9:6 was observed. F2 progeny plants of the cross between the two resistant PI’s were resistant, except for a few plants that were partially susceptible, suggesting that some of the resistance genes in PI 197088 and PI 330328 are not allelic.


Introduction
Downy mildew (DM) is a devastating foliar disease of cucurbits with a global distribution. The causal agent, Pseudoperonospora cubensis (Berk. and Curt.) Rost. (Oomycota, Peronosporaceae), is an obligate biotrophic oomycete pathogen that attacks over 40 host plant species belonging to 20 genera of the Cucurbitaceae [1,2]. Typical symptoms in cucumber consist of chlorotic irregular lesions with sporulation on the lower leaf surface. Several review articles provide basic information on the biology, epidemiology, and control of the disease [1][2][3][4][5]. Infection may take place if free leaf moisture is available for ≥2 h at an appropriate temperature [6]. The sporangia release biflagellate zoospores that swim towards the stomata where they encyst, germinate, and penetrate. Hyphae grow into the intercellular space, colonize the mesophyll tissue, and establish intracellular haustoria for nutrient uptake. Haustoria also deliver effector proteins to facilitate the establishment and/or combat the host plant's defense response system [3,7]. At ≥4 days post-inoculation, hyaline sporangiophores emerge from stomata bearing dark sporangia at their tip. Sporangia are dispersed by wind or rain and Despite the extensive screening and breeding efforts that were done to identify sources of resistance and to incorporate them into commercial cultivars [9], no cultivars currently offer a high level of resistance to the populations of P. cubensis that occur in different parts of the world. The reasons for the lack of resistant cultivars may derive from the heterozygosity of the resistant sources used for breeding, the continuous changes in the population structure of the pathogen, and the difficulty to pyramid the number of genes/QTLs in one cultivar.
The most promising current sources of resistance are PI 197088 and PI 330628 [30]. However, no data are available on the magnitude of their resistance against different isolates of P. cubensis from different parts of the world.
The objectives of this study were to: (i) stabilize PI 197088 and PI 330628 for resistance against multiple isolates of P. cubensis from different parts of the world. (ii) study the mechanism of resistance of PI 197088 and PI 330628 against P. cubensis. (iii) determine the mode of inheritance of resistance in PI 197088 and PI 330628 against multiple isolates of P. cubensis from different parts of the world.

Pathogen
Forty-four field isolates of P. cubensis that were collected during 1980-2018 from 13 countries were available to perform the different experiments in the present study (Table 2), including two F1 hybrid isolates that were produced in our laboratory by crossing A1 and A2 field isolates from different hosts as described before [2]. A subset of 23 isolates was used to screen the resistance of PI 197088 and PI 330628 while other subsets of isolates were used to determine the resistance of F2 and F3 populations. The isolates were maintained by repeated inoculation of detached cucumber leaves of the universal susceptible cucumber line Nadiojny (own bred). Long-term maintenance of the isolates was done by storing freshly-sporulating leaves in dry paper bags at −80 • C.  Table 1 in [4].

Isolate
Year

Crosses
The susceptible SMR-18 and the resistant PI 197088 and PI 330628 were self-pollinated for three generations to ensure homozygosity (see below). Crosses were made between the susceptible SMR-18 and each of the resistant PI 197088 or PI 330628. Another cross was done between these two resistant accessions. F1 plants were grown in a net-house (insect-proof) and self-pollinated to obtain F2 populations. A single fruit was harvested from each F1 plant and the F2 seeds were grown the following season in net houses and self-pollinated to obtain F3 plants.

Inoculation of Detached Leaves
Parents, F1, F2, and F3 plants were grown erect in net-houses during 2014-2019. The third leaf from the top of 15-20-leaf plants were excised, placed on a wet filter paper in flat plastic trays (60 × 40 × 5 cm), lower surface uppermost, and spray-inoculated with a sporangial suspension of P. cubensis (2000 sporangia per mL). Trays were covered with transparent plastic bags and kept for 16 h in a dew chamber at 18 • C in the dark and thereafter in a growth chamber at 20 • C (14 h light/day, 100 µmole·s −1 ·m 2 ) for 7 days.

Disease Assessment in Detached Leaves
Two readings were taken from each detached leaf at 7 days post-inoculation (dpi): the proportion of leaf area (0-100%) occupied with downy mildew lesions and the intensity of sporulation (0-3 scale) as visualized with a ×10 magnifying lens. The two values were multiplied to obtain a disease scoring scale of 0-300 ( Figure 1). Leaves showing a score of 0-20; 21-200; 201-300 were considered resistant (R); moderately resistant (MR) and susceptible (S), respectively.

Microscopy
The methods used by Cohen et al. [33] were used. Briefly, healthy and infected leaf discs were clarified in boiling ethanol, placed in aniline blue solution (0.05% aniline blue in 0.05 M K 2 HPO 4 , pH 8.9) at 4 • C for 24 h, stained with 0.01% calcofluor (Sigma) and examined with an Olympus A70 epi-fluorescent microscope. Sporangiophores on leaf surface fluoresced blue and sporangia looked dark. Fungal structures inside the leaf showed green-yellow fluorescence. Callose-encased haustoria were seen yellow. Staining for lignin was done with ethanol-clarified leaf discs. They were placed on microscope slides, treated with 2% phloroglucinol in methanol, and then with 0.25% HCl. A red color was visible in the lignified mesophyll cell.    Table 3. Nine PI 197088 plants ( Figure 4A) and three PI 330628 plants ( Figure 4B) were resistant to all 23 isolates of the pathogen, suggesting heterozygosity of the original accessions. One resistant plant of each PI was self-pollinated for two more generations and its offspring plants were all found resistant to these isolates of the pathogen. These two plants were used to study the resistance mechanisms and the mode of inheritance of resistance.  Table 2.

Microscopy of Resistance in PI 197088 and PI 330628
Leaf discs (15 mm diameter) were removed at 6 dpi from detached infected leaves (isolate 83C) of SMR-18, PI 197088, and PI 330628 and examined under UV illumination. Abundant sporangiophores with sporangia were seen in the susceptible SMR-18 ( Figure 5A-C). In contrast, a few mycelium runners bearing callose-encased haustoria were seen in PI 197088 ( Figure 5E,G). Similar callose depositions were seen in PI 330628 (not shown). Sporangiophores were partially branched with no sporangia seen in the resistant plants ( Figure 5F). Bright-field microscopy of phloroglucinol-stained infected leaf discs showed no lignin staining in SMR-18 ( Figure 5D) but heavy lignin accumulation in the resistant PI 197088 ( Figure 5H). Similar lignin accumulation was seen in the resistant PI 330628 (not shown). Resistance to downy mildew in PI 197088 and PI 330628 was stable at a colonization temperature of 14 • C (not shown).

Quantification of Resistance in Adult Plants
The third leaf from the top of adult SMR-18, PI 197088, and PI 330628 plants (n = 10) grown in a net house was detached and drop-inoculated on the lower leaf surface with isolate 260. Fifteen mm leaf discs were sampled at 7 dpi for microscopic examination of sporulation. UV-epifluorescence microscopy of calcofluor-stained leaf discs revealed 650 ± 123 sporangiophores/cm 2 in SMR-18 as against 11 ± 8 and 18 ± 12 sporangiophores/cm 2 in PI 197088 and PI 330628, respectively ( Figure 6A). The sporangiophores in SMR-18 were branched dichotomously three times, twice in PI 330628 and only once in PI 197088. The number of sporangia produced in SMR-18, PI 197088, and PI 330628 was 91 ± 5, 1 ± 0.3, and 6 ± 1 thousands of sporangia per cm 2 , respectively ( Figure 6B). Similar results were obtained with other isolates of the pathogen (not shown). The data indicated that PI 197088 is slightly more resistant to downy mildew than PI 330628. Artificial inoculation of intact plants (10-leaf stage) in the field with isolate 260 resulted at 9 dpi with the production of chlorotic lesions in SMR-18 and minute necrotic lesions in PI 197088 and PI 330628 ( Figure 7A-C). Some infected leaves were detached, placed on wet filter paper and incubated in a growth chamber at 20 • C for three days (14 h light a day, 100 µmole·s −1 ·m 2 ). At 13 dpi, SMR-18 produced the largest lesions (17.5 ± 3.5 mm) and the highest number of sporangia 71.9 ± 6.2 × 10 3 per lesion whereas PI 197088 produced the smallest lesions (1.5 ± 0.7 mm) with the lowest number of sporangia per lesion (0.4 ± 0.1 × 10 3 ) ( Figure 7G,H). The F1 hybrid plants produced intermediate-size lesions with a moderate number of sporangia per lesion ( Figure 7G,H). Percent leaf area infected at 27 dpi in fully-grown, blooming plants in the field is shown in Figure 7I. While SMR-18 exhibited 72% infected leaf area, PI197088 and PI330628 showed 1.2 and 4.4% infected leaf area, respectively. Their F1 plants were moderately infected ( Figure 7I). AUDPC values at the end of the season are shown in Figure 7J. These results indicated that resistance of 197088 and 330628 is controlled by partially dominant gene(s). When SMR-18, PI 197088, and PI 330628 (n = 10) were planted in a net house and thus exposed to natural infection, severe downy mildew developed in SMR-18 within four weeks after planting whereas no disease symptoms were seen in PI 197088 or PI 330628 (Figure 8). The disease was not visible in these two PI's even at four months after planting when they carried mature fruits (produced by hand pollination), suggesting that homozygosity may avoid the appearance of the susceptible reaction reported to occur in these accessions at an advanced stage of growth in the field [28]. Resistance to downy mildew in melon Cucumis melo PI 124111F was shown to be active at colonization temperatures of >15 • C [34] due to the expression of eR genes [35]. Here, we observed that both PI 197088 and PI 330628 sustained full resistance to multiple isolates of P. cubensis when incubated after inoculation at either 14 • C or 20 • C (detached leaf bioassay), suggesting that unlike melon, expression of resistance against downy mildew in cucumber occurs at a low temperature of 14 • C.

Inheritance of Resistance in Detached Leaves of Adult Plants
A set of 22 isolates was used to inoculate F2 plants of the cross PI 197088 × SMR 18 and a set of 14 isolates (all included in the former set) was used to inoculate F2 plants of the cross PI 330628 × SMR 18. Graphical illustrations of the disease scores are given for only 10 and 8 isolates of the above respective crosses (Figures 9 and 10). Full numerical scores for all 22 and 14 isolates are given in Table 4. Data in Figures 9 and 10 show a unique segregation pattern for each isolate. With some isolates, a similar pattern was seen for F2 of both crosses PI 197088 × SMR 18 and PI 330628 × SMR 18. Large differences in the response to inoculation were observed between plants, depending on the isolate used for inoculation. A single plant could react with different scores when inoculated with different isolates. The Mendelian analyses of the data are presented in Table 4. When plants' responses were classified into two categories, F2 plants of the cross PI 197088 × SMR-18 showed five segregation ratios, depending on the isolate used for inoculation: 3:1 (2 isolates), 1:3 (11 isolates), 1:15 (6 isolates), 9:7 (2 isolates), and 13:3 (one isolate) (Table 4A). When three categories were used for classification, only 2 isolates out of 22, obeyed the Mendelian segregation of 9:6:1 (Table 4A). No inheritance model could be assigned to the results obtained with the other 19 isolates. Five segregation ratios were observed when two categories were applied for classification of the F2 plants of the cross PI 330628 × SMR-18: 3:1 (1 isolate), 1:3 (8 isolates), 1: 15 (2 isolates), 7:9 (2 isolates), and 9:7 (1 isolate) (Table 4B). When three categories were used for classification, only one isolate out of 14 obeyed a segregation ratio of 9:7. No inheritance model could be assigned to the results obtained for the other isolates (Table 4B).

Inheritance of Resistance in Intact Field-Grown Plants
The response of the parents F1 and F2 plants to downy mildew in the field in 2019 is shown in Figure 11. A continuous response pattern to the disease was observed in F2 plants of PI 197088 × SMR-18 ( Figure 11A,B) and of PI 330628 × SMR-18 ( Figure 11D,E). Both resistant parents were completely resistant all along the season (until fruit maturity) whereas F1 plants were moderately resistant ( Figure 11C,F).  Table 5 summarize the segregation for the resistance of F2 plants in the field during 2013-2019. F2 plants of the cross PI 197088 × SMR-18 were tested in six seasons whereas F2 plants of the cross PI 330628 × SMR-18 were tested in two seasons. When two categories were used to classify the response of the plants to the disease (R and S)" two segregation ratios were observed, 1:15 or 1:63. When three categories were used for classification (R, MR, and S), two segregation ratios were observed, 1:14:1 or 1:9:6. The data suggest that genetic control of resistance in F2 plants varies between seasons, probably depending on the isolate prevailing in the field at each season.

Resistance in F2: F3 Plants
Two field-grown resistant F2 plants (107 and 111) of the cross PI 197088 × SMR-18, were tested (detached leaf bioassay) for resistance against 14 isolates of P. cubensis. Plant 107 was resistant to all isolates, except to the hybrid isolate 83C × 98P, while plant 111 was resistant to all isolates except to the hybrid isolate 172 B × 183 C (Table 6A), suggesting enhanced virulence of hybrid isolates. Each plant was self-pollinated and detached leaves from the F3 plants growing in the field were inoculated with each of the four isolates of the pathogen. The segregation data are shown in Table 6B. F3 plants segregated into R: S at a ratio of 3:1, 1:3, 1:15, or 7:9, depending on the isolate used for inoculation. No genetic model fits the segregation data when three response categories were used because no MR-scored plants were detected in the progenies (Table 6B).

Resistance of F3 Plants in the Field
Seventy-nine F2 plants of the cross PI 197088 × SMR-18, six resistant (score 0), and 73 susceptible (score ≥ 200) ( Figure 10) were self-pollinated to produce F3 seeds. Ten plants of each F3 entry were transplanted to the field and exposed to natural infection. Disease records were taken at weekly intervals for 22 days after the onset of the disease. Mean AUDPC and SD values for each F3 entry (n = 10) are shown in Figure 12. Mean AUDPC of SMR-18, PI 197088, and their F1 were 635, 10, and 206, respectively. F3 entries derived from resistant F2 plants showed AUDPC values ranging from 62 to 293 and those derived from susceptible F2 plants showed AUDPC values ranging from 31-545 ( Figure 12). The results indicate that F3 plants are heterozygous for resistance, regardless of whether they were derived from a resistant or a susceptible F2 plant.

Discussion
The population of P. cubensis in the field may consist of many isolates, pathotypes, or races with varying degrees of pathogenicity or virulence thus rendering host resistance ineffective. Indeed, combating downy mildew (DM) in cucumber through host plant resistance or fungicide applications has become more complex in the past two decades due to the emergence of new pathotypes, races, and mating types of the causal agent P. cubensis. Old cucumber cultivars resistant to DM succumbed to the new pathotypes, and the old fungicidal chemistries lost activity due to the prevalence of resistant isolates of the pathogen [4,13]. Breeding cucumber for DM resistance is a long and laborious task due to the lack of stable, multi-race resistant sources and the complex mode of resistance inheritance.
Here we identified two sources of wild cucumber with multi-race/pathotype resistance. We characterized the mechanism of their resistance and determined the way they inherit resistance to their progeny plants. Because the resistance of accession to a local isolate of P. cubensis does not necessarily mean that it will be resistant to isolates that prevail in other locations, we used a large collection of isolates from different parts of the world to screen resistance. We developed a detached leaf bioassay in which we could determine the resistance of a single plant to multiple isolates of P. cubensis. We thus were able, for the first time, to study the mode of inheritance of resistance to multiple isolates and predict the performance of the resistant pedigrees in other countries.
Of the six Cucumis sativum genotypes known to exhibit resistance against P. cubensis [4], only PI 197088 and PI 330628 [28,30] exhibited multiple-isolate resistance. They were self-pollinated for three generations to bring their multiple-isolate resistance to homozygosity. The stabilized lines were used for the inheritance studies reported here.
When grown in the field under natural epiphytotic conditions, no disease was observed on the leaves of PI 197088 or PI 330628. However, when artificially inoculated in the field or in growth chambers, a few necrotic lesions did appear. Microscopic observations revealed that PI 197088 and PI 330628 exhibit similar responses to artificial inoculation with P. cubensis. The pathogen ceased developing at a relatively late stage after penetration and developed some initial hyphae and haustoria. The haustoria formed were encased with callose, which probably inhibits the intake of nutrients into the mycelium, while the infected cells accumulated lignin-like, phloroglucinol-positive materials. A similar structural mode of resistance was observed in melons resistant to P. cubensis [33,34]. These defense compounds still allowed some deteriorated sporangiophores to emerge from the stoma but almost totally prevented sporangial production. We show here that unlike the resistance in melon which breaks down at 14 • C [34], the resistance in PI 197088 and PI 330628 remained effective at a low colonization temperature of 14 • C.
We used a double visual scoring system (percent infected leaf area and sporulation intensity) to determine the level of resistance to DM in detached leaves ( Figure 1). We observed that leaves taken from F2 plants of the cross PI 197088 × SMR-18 or 330628 × SMR-18 segregated in their phenotypic responses to infection with P. cubensis ranging from complete resistance to high susceptibility. The pattern of segregation depended on the isolate used for inoculation. The differential pattern of response to different isolates indicated that a number of genes might be involved in resistance. The Mendelian analysis was employed to the segregated populations after categorical classification into S: R or R: MR: S. The analysis of two categories R and S indicated that resistance in PI 197088 or PI 330628 is controlled by either 1 dominant, 1 recessive, or 2 recessive genes, depending on the isolate used for inoculation. Analysis with three categories of S, MR, and R did not fit, in most cases, any  [28].
Isolate-dependent inheritance of disease resistance is a rarely reported phenomenon [36]. Lapin et al. [37] showed that unlike most natural Arabidopsis thaliana accessions that are susceptible to one or more isolates of the downy mildew pathogen Hyaloperonospora arabidopsidis, accession C24 is resistant to all isolates tested. The resistance of C24 was found to be a multigenic trait with complex inheritance. Many identified resistance loci were isolate-specific and located on different chromosomes. Among the C24 resistance QTLs, there were dominant, codominant, and recessive loci. Interestingly, none of the identified loci significantly contributed to resistance against all three tested isolates.
Unlike wild cucumbers, resistance of the wild melon (Cucumis melo L) PI 124111F against P. cubensis is broad-spectrum but not isolate-specific [38]. That resistance was controlled genetically by two partially dominant, complementary loci [39]. Unlike other plant disease resistance genes, which confer an ability to resist infection by pathogens expressing corresponding avirulence genes, the resistance of PI 124111F to P. cubensis is controlled by enhanced expression of the enzymatic resistance (eR) genes At1 and At2. These constitutively expressed genes encode the photorespiratory peroxisomal enzyme proteins glyoxylate aminotransferases. The low expression of At1 and At2 in susceptible melon lines is regulated mainly at the transcriptional level. This regulation is independent of infection with the pathogen. Transgenic melon plants overexpressing either of these eR genes displayed the enhanced activity of glyoxylate aminotransferases and remarkable resistance against P. cubensis [35,40]. Our attempts to transfer At1 and At2 to cucumber did not succeed (Cohen, unpublished data).
The results presented here corroborate with other studies in which multiple QTLs for resistance against P. cubensis were identified in PI 197088 and PI 330628. (Table 1). Wang et al. [30] reported QTL mapping results for DM resistance with F2:3 families from the cross between DM-resistant inbred line PI 330628 (WI7120) and susceptible '9930'. Four QTLs, dm2.1, dm4.1, dm5.1, and dm6.1 were consistently and reliably detected across at least three of the four environments which together could explain 62-76% phenotypic variations. Among them, dm4.1 and dm5.1 were major effect QTL and dm2.1 and dm6.1 had moderate and minor effects, respectively.
Wang et al. [28] used recombinant inbred lines from a cross between PI 197088 and the susceptible line 'Coolgreen'. Phenotypic data on responses to natural DM infection were collected in three years and five locations from replicated field trials in North Carolina. The observed ratings followed a normal distribution that covered a large range of ratings at each environment and date. The interaction effects of genotype-by-location and genotype-by-year were significant at all ratings. QTL analysis identified 11 QTL for DM resistance harbored on chromosomes 1-6, accounting for more than 73.5% total phenotypic variance. Among the 11 DM resistance QTLs, dm5.1, dm5.2, and dm5.3 were major effect contributing QTL whereas dm1.1, dm2.1, and dm6.2 conferred susceptibility. The QTL dm4.1 which had a moderate effect was likely the same as the major-effect QTL dm4.1 detected in PI 330628 [30]. Three DM QTLs dm2.1, dm5.2, and dm6.1, were co-localized with powdery mildew (PM) QTLs, pm2.1, pm5.1, and pm6.1, respectively, which was consistent with the observed linkage of PM and DM resistances in PI 197088.
Katz et al. [29] reported on nine QTLs associated with resistance of PI 197088 against each of the seven isolates of P. cubensis. They examined for two years the response of a segregating F2 family (PI-197088 × SMR-18, n = 170) to seven isolates in growth chambers and the field. NGS (Next-Generation Sequencing) was performed for genotyping, and polymorphic SNPs were obtained from the same populations in both years. QTLs obtained for isolate 23C-resided on chromosomes 4 and 5; for isolate Pol.1-on chromosomes 1, 4, and 5; for isolate Pol.4-on chromosome 7; for isolate US-506on chromosomes 1 and 2; for isolate 81C-on chromosomes 4 and 5; for isolate 88C-on chromosomes 3 and 6; for isolate 90C-on chromosomes 1, 4, and 6; for field isolate 2016, on chromosomes 3 and 5, and for field isolate 2017-on chromosomes 4 and 5. These authors concluded that the inheritance of resistance against DM in PI 197088 was isolate-dependent.
Tian et al. [41] sequenced 14% of the genome of one isolate of P. cubensis and identified 32 putative RXLR effector proteins and 29 secreted peptides with high similarity to RXLR effectors. They suggested that these effectors might play pivotal roles in pathogen fitness and pathogenicity. Sexual reproduction of the pathogen [12,42] may result in recombinant isolates which carry various combinations of effector proteins. It might, therefore, occur that isolates evolved in different parts of the world and therefore belong to different races, pathotypes, and mating types, each carries a unique set of effectors. Of this set of effectors, some might be secreted while others may not. Of the secreted effectors, some may recognize certain R genes in the host while others will not. This will make some host genotypes resistant to some genotypes of the pathogen.
The isolate-dependent inheritance of resistance of cucumber against P. cubensis may indicate that each isolate secretes a different battery of effectors that ignite a unique set of R genes in the host, thus making the inheritance of resistance isolate-dependent.