Avia E. Rubin

Mechanisms of induced resistance in lettuce against Bremia lactucae by DL-β-amino-butyric acid (BABA)

BABA induced local and systemic resistance in lettuce (Lactuca sativa) against the Oomycete Bremia lactucae. Structure-activity analysis showed no induced resistance by related amino-butanoic acids or β-alanine. The R-enantiomer of BABA induced resistance whereas the S-enantiomer did not, suggesting binding to a specific receptor. Other compounds known to be involved in SAR signaling, including abscisic acid, methyl-jasmonate, ethylene, sodium-salicylate and Bion® (BTH) did not induce resistance. Systemic translocation of 14C-BABA and systemic protection against downy mildew were tightly correlated. BABA did not affect spore germination, appressorium formation, or penetration of B. lactucae into the host. Epifluorescence and confocal microscopy revealed that BABA induced rapid encasement with callose of the primary infection structures of the pathogen, thus preventing it from further developing intercellular hyphae and haustoria. Invaded host cells treated with BABA did not accumulate phenolics, callose or lignin, or express HR. In contrast, cells of genetically-resistant cultivars accumulated phenolics, callose and lignin and exhibited HR within one day after inoculation. The callose synthesis inhibitor DDG did not inhibit callose encasement nor compromised the resistance induced by BABA. PR-proteins accumulated too late to be responsible for the induced resistance. DAB staining indicated that BABA induced a rapid accumulation of H2O2 in the penetrated epidermal host cells. Whether H2O2 stops the pathogen directly or via another metabolic route is not known.

Mutagenesis of Phytophthora infestans for Resistance Against Carboxylic Acid Amide and Phenylamide Fungicides

The carboxylic acid amide (CAA) fungicides mandipropamid, dimethomorph, iprovalicarb, and the phenylamide fungicide mefenoxam (MFX, the active enantiomer of metalaxyl) are anti-oomycete fungicides effective against downy mildews and late blight. Resistance against MFX was reported in nature in several oomycetes including Phytophthora infestans and Plasmopara viticola, whereas resistance against CAAs was reported in P. viticola but not in P. infestans. In this study the mutability of P. infestans for resistance against CAAs and MFX (as a control) was explored under laboratory conditions. UV light or chemical mutagens (e.g., ethyl methan sulfonate [EMS]) were applied to sporangia, and the emergence of mutants resistant to CAAs or MFX, or with altered mating type, was followed. Many mutants resistant to CAAs developed at generation 0 after mutagenesis, but all showed erratic, instable resistance in planta, diminishing after 1 to 8 asexual infection cycles, and failed to grow on CAA-amended medium. In contrast, 19 mutants resistant to MFX were obtained: 6 with UV irradiation (in isolates 28 or 96) and 13 with EMS (in isolates 408, 409, and 410). In three experiments, a shift in mating type, from A1 to A2, was detected. To elucidate whether or not resistance to CAAs is recessive and therefore might emerge only after sexual recombination, A1 and A2 mutants were crossed and the F1 and F2 progeny isolates were tested for resistance. Offspring isolates segregated for resistance to MFX, with resistant isolates maintaining stable resistance in vitro and in planta, whereas all progeny isolates failed to show stable resistance to CAAs in planta or in vitro. The data suggest that P. infestans could be artificially mutated for resistance against MFX, but not against CAAs.

Light Suppresses Sporulation and Epidemics of Peronospora belbahrii

Peronospora belbahrii is a biotrophic oomycete attacking sweet basil. It propagates asexually by producing spores on dichotomously branched sporophores emerging from leaf stomata. Sporulation occurs when infected plants are incubated for at least 7.5h in the dark in moisture-saturated atmosphere at 10-27°C. Exposure to light suppresses spore formation but allows sporophores to emerge from stomata. Incandescent or CW fluorescent light of 3.5 or 6 µmoles.m2.s-1 respectively, caused 100% inhibition of spore formation on lower leaf surface even when only the upper leaf surface was exposed to light. The inhibitory effect of light failed to translocate from an illuminated part of a leaf to a shaded part of the same leaf. Inhibition of sporulation by light was temperature-dependent. Light was fully inhibitory at 15-27°C but not at 10°C, suggesting that enzyme(s) activity and/or photoreceptor protein re-arrangement induced by light occur at ≥15°C. DCMU or paraquat could not abolish light inhibition, indicating that photosystem I and photosystem II are not involved. Narrow band led illumination showed that red light (λmax 625 nm) was most inhibitory and blue light (λmax 440 nm) was least inhibitory, suggesting that inhibition in P. belbahrii, unlike other oomycetes, operates via a red light photoreceptor. Nocturnal illumination of basil in the field (4-10 µmoles.m2.s-1 from 7pm to 7am) suppressed sporulation of P. belbahrii and reduced epidemics of downy mildew, thus reducing the need for fungicide applications. This is the first report on red light inhibition of sporulation in oomycetes and on the practical application of light for disease control in the field.

First Report of the Occurrence and Resistance to Mefenoxam of Peronospora belbahrii, Causal Agent of Downy Mildew of Basil (Ocimum basilicum) in Israel

Downy mildew in basil was first reported from Uganda in 1933 (4). In 2004, it was reported from Italy (3) and, thereafter, from other countries around the world. In Israel, the disease was first observed in November 2011 in two greenhouses located in the northern part of the Jordan Valley. Within a month, second and third outbreaks of the disease occurred simultaneously near the southwest and southeast borders of Israel, 250 km from the initial disease outbreak. By the summer of 2012, the disease had appeared throughout the country, causing major economic damage. The causal agent, identified as Peronospora belbahrii (see below), produced chlorotic lesions on leaf blades with sporangia developing on the lower leaf surfaces. Lesions gradually turn necrotic, and infected leaves abscised. Sporangia were dark purple, oval, 30.4 ± 2.9 μm long × 21.4 ± 1.7 μm wide. Sporangiophores emerged from stomatal openings in a saturated atmosphere, were hyaline, 400 to 600 μm long, dichotomously branched, with three to five branches per sporangiophore, and bore a single sporangium on each branchlet tip. Oospores, seldom seen, were brown, round, and 46.2 ± 2.8 μm in diameter. Sporangia germinated directly, each producing a single germ tube that penetrated the periclinal wall of epidermal cells. PCR assays using sporangia and infected leaves as the template, and specific BAZ primers (1), produced a 134-bp band typical of P. belbahrii (1,2). Twenty isolates, collected from 12 locations in Israel from December 2011 to September 2012, were all sensitive to mefenoxam as the isolates did not cause symptoms on 15-leaf, potted basil plants (cv. Peri, Volcani Center, Israel) that were sprayed with 10 μg mefenoxam/ml (Ridomil Gold 48%, Syngenta, Basel, Switzerland) prior to inoculation. However, one isolate collected in early October 2012 from a severely infected plant in a greenhouse at Rehov in Bet-Shaan Valley, in which the plants had been treated with mefenoxam, was resistant to mefenoxan, showing abundant sporulation on leaves of potted basil plants that had been sprayed with 1,000 μg of mefenoxam/ml prior to inoculation. To our knowledge, this is the first report of the occurrence of downy mildew in basil in Israel. This is also the first global report of resistance to mefenoxam in P. belbahrii. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) R. Djalali et al. Mycol. Progress 11:961, 2012. (3) A. Garibaldi et al., Plant Dis. 89:683, 2004. (4) C. G. Hansford. Rev. Appl. Mycol. 12:421, 1933.

Formation and Infectivity of Oospores of Pseudoperonospora cubensis, the Causal Agent of Downy Mildew in Cucurbits

The oomycete Pseudoperonospora cubensis attacks members of the Cucurbitaceae, causing severe foliage damage especially to cucumber and melon. Recently, new pathotypes of this oomycete appeared in Israel (2) and Italy (1) and highly aggressive isolates appeared in the United States (3). Since oospores of P. cubensis were rarely seen and sexual propagation by oospores was never reported (4), it is assumed that it propagates clonally by sporangia. Here we report on sexual reproduction of P. cubensis under controlled conditions in the laboratory. We found that field isolates belonging to the old pathotype 3 or to the new pathotype 6 (2) inoculated singly onto detached leaves of cucurbits in growth chambers at 15 or 20°C produced no oospores, even after prolonged incubation periods. However, when sporangia of some paired field isolates were mixed together at a 1:1 ratio, similarly inoculated onto detached leaves, and incubated at 15 or 20°C, numerous oospores (up to ~300/cm2) were formed in the mesophyll within 6 to 11 days, depending on the isolates pair, the host inoculated, and temperature. Oospores were also formed at 12.5°C but not at 25°C. Oospores developed in intact plants when kept at 15 or 20°C under a humidity-saturated atmosphere during disease development. Oospores were round, light brown to brown with an average diameter of ~40 μm. Oospores were produced in Cucumis sativum (cvs. Nadiojni and Dalila) and Cucumis melo (cvs. Ananas-Yokneam and Ein-Dor) but not in Cucurbita pepo (cv. Arlika, Beiruti), C. moschata (cv. Dalorit), or C. maxima (cv. Tripoli). To verify that oospores are infective, cucumber or melon leaves containing oospores were homogenized in water. The homogenate was twice brought to dryness at 25 to 30°C in petri dishes to differentially kill the vegetative structures of the pathogen (sporangia, cystospores, zoospores, and mycelia), resuspended in water, and inoculated onto detached leaves of various cucurbits in growth chambers at 15 or 20°C. Downy mildew lesions carrying sporangia appeared within 7 to 20 days in leaves of Cucumis sativum, Cucumis melo, and C. moschata but not in C. pepo or C. maxima. The recombinant origin of the F1 offspring isolates was confirmed by mefenoxam sensitivity tests, random amplified polymorphic DNA, and simple sequence repeat analyses. F1 progeny isolates of some crosses lost pathogenicity to C. moschata or C. maxima, toward which one of their parents was pathogenic, while others gained pathogenicity to Luffa cylindrica or Citrullus lanatus toward which neither parent was pathogenic. Data confirmed that isolates of P. cubensis can mate to produce oospores, especially under constant humidity conditions; such oospores are infective to cucurbits and F1 progeny isolates show altered sensitivity to fungicides or altered host range relative to their parents. To our knowledge, this is the first report of oospore formation by P. cubensis in the laboratory and on their pathogenicity to cucurbits. Reasons for the parallel appearance of new pathotypes of P. cubensis in Israel in 2002 (2) and Italy in 2003 (1) and the reemergence of highly aggressive isolates of the pathogen in the United States in 2004 (3) are not known. They may be related to oospore production and sexual recombination in P. cubensis. References: (1) C. Cappelli et al. Plant Dis. 87:449, 2003. (2) Y. Cohen et al. Phytoparasitica 31:458, 2003. (3) G. J. Holmes et al. Am. Veg. Grower. February, 14-15, 2006. (4) A. Lebeda and Y. Cohen. Eur. J. Plant Pathol.129:157, 2011.

Seed transmission of Pseudoperonospora cubensis.

Pseudoperonospora cubensis, an obligate biotrophic oomycete causing devastating foliar disease in species of the Cucurbitaceae family, was never reported in seeds or transmitted by seeds. We now show that P. cubensis occurs in fruits and seeds of downy mildew-infected plants but not in fruits or seeds of healthy plants. About 6.7% of the fruits collected during 2012–2014 have developed downy mildew when homogenized and inoculated onto detached leaves and 0.9% of the seeds collected developed downy mildew when grown to the seedling stage. This is the first report showing that P. cubensis has become seed-transmitted in cucurbits. Species-specific PCR assays showed that P. cubensis occurs in ovaries, fruit seed cavity and seed embryos of cucurbits. We propose that international trade of fruits or seeds of cucurbits might be associated with the recent global change in the population structure of P. cubensis.

Pathogenic Fitness of Oosporic Progeny Isolates of Phytophthora infestans on Late-Blight-Resistant Tomato Lines

Four A1 field isolates and one A2 field isolate of Phytophthora infestans were crossed to produce oospores in tomato leaves. The oospores were extracted and mixed with perlite and water, and healthy tomato leaves were used as bait for oospore-progeny infection. Twenty-nine lesions were obtained from the four crosses and 283 single-sporangium isolates were recovered and tested on four tomato differential lines carrying different major genes (Ph-0, Ph-1, Ph-2, and 3707) for late blight resistance. The pathogenic fitness (number of sporangia per unit leaf area) of parental and progeny isolates was strongly dependent on the host genotype; it decreased in the order Ph-0 > Ph-1 > Ph-2 > 3707. The A2 parent had a higher pathogenic fitness than the A1 parents on Ph-0 and Ph-1 but similar, lower fitness on Ph-2. Different levels of pathogenic fitness were observed across all isolates on Ph-0, although Ph-0 lacks resistance genes. Pathogenic fitness on one tomato genotype was not related to the pathogenic fitness on the other tomato genotypes. Some isolates exhibited reduced pathogenic fitness relative to the respective A1 parent, whereas others demonstrated a higher pathogenic fitness compared with the A2 parent. The tomato genotype Solanum pimpinellifolium L3707/5 was resistant to all five parental isolates of P. infestans. However, 37 of the 283 progeny isolates from 11 different lesions had compatible reactions with this line, producing up to 31 × 103 sporangia/cm2. Overall, reduced fitness was more frequent among the progeny isolates than increased fitness.

Host Preference of Mating Type in Pseudoperonospora cubensis, the Downy Mildew Causal Agent of Cucurbits

The A2 mating type of Pseudoperonospora cubensis was first discovered in Israel in May 2010 on butternut gourd (Cucurbita moschata) (1). We monitored the occurrence of the A2 mating type of P. cubensis in isolates collected during May 2010 through September 2012 from downy mildew-infected cucurbit crops growing along the coastal plain of Israel. Mating type was determined by oospore production in melon leaf discs co-inoculated with sporangia of a test isolate mixed with sporangia of A1 or A2 tester isolates (2). The A1 and A2 tester isolates were maintained at 14°C (14 h light/day) by repeated inoculation of detached leaves of cucumber and pumpkin, respectively. The 29 isolates that were collected from cucumber (Cucumis sativum) were all A1. Of the 33 isolates collected from pumpkin (Cucurbita maxima), squash (C. pepo), or butternut gourd (C. moschata), 88% were A2 and 12% were A1. The host preference of mating type in P. cubensis was monitored at Bar-Ilan University farm during April to July 2012, among about 800 plants of eight cucurbit species (~100 plants per species) that were grown side-by-side in three adjacent net-houses (two 6 × 50 m and one 6 × 100 m) and exposed to natural infection. Downy mildew developed on cucumber, melon, pumpkin, squash, and butternut gourd, but not on watermelon, sponge gourd (Luffa cylindrica), or Momordica balsamina. Three-hundred and three isolates of P. cubensis were collected and tested for mating type: 123 from cucumber, 53 from melon, 30 from pumpkin, 48 from butternut gourd, and 41 from squash. The cucumber isolates expressed A1, A2, and A1A2 at a ratio of 94.3%, 3.3%, and 2.4%, respectively; the melon isolates 58.5%, 26.4%, and 15.1%; the pumpkin isolates 0%, 96.7%, and 3.3%; the butternut isolate 7.3%, 87.3%, and 5.5%; and the squash isolates 2.4%, 97.6%, and 0%, respectively. A1A2 isolates produce oospores when crossed with either A1 or A2 tester isolates. This is the first evidence suggesting a preference of A1 isolates to Cucumis spp. and of A2 isolates to Cucurbita spp. similar preference was recently observed among Chinese isolates of this pathogen (unpublished data). The mechanism(s) controlling this preference is not known. Classical genetics is currently employed to P. cubensis in order to understand if it derives from true linkage. The practical implication for downy mildew management is that growing cucumber/melon in close proximity to pumpkin/squash/butternut gourd should be avoided as it may enhance oospore production in nature. Oospores in soil were recently shown to serve as a primary source of downy mildew infection in cucumber (3). References: (1) Y. Cohen, A. E. Rubin, and M.Galperin. Plant Dis. 95:874, 2011; (2) Y. Cohen and A. E. Rubin. Eur. J. Plant Pathol. 132:577, 2012; (3) Y. J. Zhang et al. J. Phytopathol. 160:469, 2012.

Daytime Solar Heating Controls Downy Mildew Peronospora belbahrii in Sweet Basil.

The biotrophic oomycete Peronospora belbahrii causes a devastating downy mildew disease in sweet basil. Due to the lack of resistant cultivars current control measures rely heavily on fungicides. However, resistance to fungicides and strict regulation on their deployment greatly restrict their use. Here we report on a ‘green’ method to control this disease. Growth chamber studies showed that P. belbahrii could hardly withstand exposure to high temperatures; exposure of spores, infected leaves, or infected plants to 35-45°C for 6-9 hours suppressed its survival. Therefore, daytime solar heating was employed in the field to control the downy mildew disease it causes in basil. Covering growth houses of sweet basil already infected with downy mildew with transparent infra-red-impermeable, transparent polyethylene sheets raised the daily maximal temperature during sunny hours by 11-22°C reaching 40-58°C (greenhouse effect). Such coverage, applied for a few hours during 1-3 consecutive days, had a detrimental effect on the survival of P. belbahrii: killing the pathogen and/or suppressing disease progress while enhancing growth of the host basil plants.

Control of cucumber downy mildew with novel fungicidal mixtures of Oxathiapiprolin

The efficacy of four oxathiapiprolin (OXPT)-based, novel fungicidal mixtures against downy mildew in cucumber caused by Pseudoperonospora cubensis was examined in growth chambers. OXPT+chlorothalonil (CHT) mixed at weight ratio of 1 + 66.7; OXPT+ azoxystrobin (AZ) 1 + 10.3; OXPT+mandipropamid (MPD) 1 + 8.3; and OXPT+mefenoxam (MFX) 1 + 3, were compared with each other and with individual components. Mixtures performed better than all fungicides alone except for oxathiapiprolin. Of the four mixtures, OXPT+MFX outperformed the other treatments with the highest preventive, curative, translaminar, root treatment and seed treatment efficacies. Deployment in the field of such mixtures with reduced doses of oxathiapiprolin may lower the selection pressure imposed on P. cubensis and delay the buildup of subpopulations resistant to oxathiapiprolin.