Abstracts of Presentations at the 35th Congress of the Israeli Phytopathological Society

INVITED LECTURES

Transgenerational inheritance of small RNAs in Caenorhabditis elegans

O. Rechavi

Dept. of Neurobiology & Sagol School of Neuroscience, The Life Sciences Faculty, Tel-Aviv University, Ramat Aviv 69978, Israel [^* e-mail: odedrechavi@gmail.com]

The inheritance of acquired characteristics is a topic of long-standing interest and controversy. While some of the classic Lamarckian ideas have been dismissed, more recent studies suggest that certain traits acquired by an animal during its lifetime may be transmitted to future generations. Our results showed that C. elegans can inherit acquired antiviral resistance through transgenerational transmission of small RNAs, which mediate RNA interference. Antiviral RNAs (viRNAs), which protect the worm from viral propagation, can be passed down to many ensuing generations in a non-Mendelian manner, in the absence of their DNA template, and thus protect (“vaccinate”) RNAi-deficient progeny from viral propagation. Most genes are regulated by different endogenous regulatory small RNA species, and therefore inheritance of small RNA might affect the inheritance of many traits. We are currently exploring the possibility that inherited small RNAs reflect the ancestral environment.

Cloning a pair of resistance genes in melon: Molecular basis of ‘gene for gene’ responses

R. Perl-Treves*, I. Kovalski, M. Normantovich, G. Miller and A. Amitzur

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel [*e-mail: rafi.perl@gmail.com]

Using molecular markers, we have mapped the genomic locus for melon resistance towards two pathogens: Fusarium oxysporum f.sp. melonis races 0 and 1, and Papaya ring spot virus (PRSV). High resolution mapping and chromosome walking enabled the identification and cloning of a pair of genes that individually control the two resistances. The two gene sequences encode proteins of the NBL (nucleotide binding site–leucine rich repeat) family. Proteins of this large and diverse family act as receptors of the plant immune system, responsible for sensing specific pathogens in the ‘gene for gene’ interaction. We have investigated allelic polymorphism in the locus and the genes' expression patterns. Positional cloning of these two genes raises exciting questions about pathogen recognition by the respective proteins, the possible relations between these two adjacent genes and between multiple splice-variants of their transcripts. These will be considered in light of the extensive front-line research on NBL proteins, their function and expression, in model systems.

DISEASE CONTROL

Control of downy mildew in basil by nocturnal illumination

Y. Cohen^*, M. Vaknin, Y. Ben-Naim and A. E. Rubin

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel [^* e-mail: ycohen@mail.biu.ac.il]

Downy mildew in basil was first reported in Israel in December 2011. The causal agent, the biotrophic oomycete Peronospora belbahrii, propagates asexually by producing spores on the lower leaf surface. Sporulation occurs when infected plants are incubated for at least 7.5 h in the dark in a moisture-saturated atmosphere at 10–27°C. Exposure to light suppresses spore formation but allows sporophores to emerge from stomata. Incandescent or cool white fluorescent light of 3.5 or 6 μmol.m^-2.s^-1, respectively, caused 100% inhibition of spore formation. Inhibition of sporulation by light was temperature-dependent. Light was fully inhibitory at 15–27°C, but not at 10°C. The inhibitory effect of light failed to translocate from an illuminated part of a leaf to a shaded part of the same leaf. DCMU or Paraquat could not abolish light inhibition, indicating that Photosystem I and Photosystem II are not involved. Narrow-band light emitting diode 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. This inhibitory effect of light on sporulation was utilized for disease control at Bar-Ilan University Farm. Nocturnal illumination of basil grown during May–July 2013 in net-houses (4–10 μmol.m-².s^-1 from 7 p.m. to 7 a.m.) suppressed sporulation of P. belbahrii and reduced epidemics of downy mildew, thus lessening the need for fungicide applications. Disease control was further improved when fungicides and illumination were combined. However, similar experiments conducted during October–November 2013 were unsuccessful, as light did not inhibit sporulation due to cool night temperatures. The technology of nocturnal illumination is currently being adopted by basil growers in Israel to control downy mildew.

Low efficacy of systemic fungicides against grape powdery mildew and measures to improve disease control

S. Ovadia^1,*, O. Frenkel² and M. Reuveni³

¹ Carmel Mizrahi Wineries, Rishon leZiyyon 75100 [^* e-mail: shmova@zahav.net.il]; ² Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250; and ³ Golan Research Institute, University of Haifa, Qazrin 12900, Israel

Powdery mildew of grapes caused by Erysiphe necator Schwein is one of the most widespread and destructive diseases in vineyards in all viticultural regions. The fungus attacks the green parts of the vine and causes much damage to clusters and yield. Disease control is based on an epidemiological study and an effective Decision Support System, “Eshkol”, which enables a significant reduction in number of fungicide applications. In the last decade low efficacy in disease control was observed due to foliar applications of either DMI (in 2004–06) or of strobilurin fungicides (from 2007). The observations in vineyards were also tested in a large number of field trials for the efficacy of the strobilurins and to develop appropriate measures to improve disease control. The results showed that disease incidence following strobilurin application was relatively high and was similar to the untreated control plots, up to 90–100%, while disease severity was relatively low. The powdery mildew symptoms that developed on these treated plots seemed to have a different phenotype. Our findings showed that use of tank mixes of fungicides of different modes of action during the sensitive phenological stage from bloom to berries to the beginning of the ‘touch’ stage improved disease control. Efficacy was dependent on the type of fungicides used in mixtures, their order during applications and on early applications of fungicides before bloom. A study to examine the genetic variations of isolates collected from various plots, including those of different phenotype, has recently been initiated. So far, there is no indication for an establishment of isolates from new genetic groups in our region. This study is in progress.

‘OrganoCide’ – a new organic fungicide

A. Ovadia^1,* and Y. Gesundhait²

¹ Agronomia - Agricultural Services (2001) Ltd., Gedera 70750 [^* e-mail: amos@agronomia.co.il]; and ² BotanoCap Ltd., Ashqelon 78172, Israel

OrganoCide is a new product containing oregano plant extract oil. It is a known kitchen spice plant generally used in the Mediterranean and particularly in Italy. It is recognized as a medicinal plant and is used for disinfection against bacteria and fungi in wound healing and against skin irritation. Oregano also improves indigestion, cough vomiting and more. In our product, OrganoCide, oregano is formulated through a novel microencapsulation technology and is organic farming certified. The microencapsulation extends and controls the availability of the oil, reduces phytotoxic effects and protects it from light degradation, hydrolysis and decomposition by microorganisms. OrganoCide is licensed and indicated for the prevention of Sclerotinia in carrots in postharvest. In addition, many field tests are now being conducted to extend the label of the product. For example, Botrytis is sensitive to OrganoCide when sprayed on cucumbers, tomatoes and grapes. Powdery mildew is sensitive to OrganoCide when sprayed on grapes and cucumbers. When applied to the soil it is very effective in controlling Rhizoctonia and powdery scab in potatoes. The active substance is volatile. Currently efficacy tests are being performed to control fungi through an atmospheric delivery system. For the time being, we have identified activity in reducing Botrytis, Penicillium and Colletotrichum. The product is safe when applied to the soil in doses of up to 20 liters per hectare for all tested crops. Foliage spray and storage applications have identified sensitive crops and other crops where the application was found safe. BotanoCap is currently studying and testing the activity of other natural materials for its microencapsulation technology.

Solar disinfection of water for controlling plant and human pathogens

E. Zeira^1,2,*, Y. Helman¹ and A. Gamliel²

¹ Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100 [*e-mail: eyal.zeira@mail.huji.ac.il]; and ² Dept. of Agricultural Engineering, ARO, The Volcani Center, Bet Dagan 50250, Israel

The uncertain availability of fresh water during recent decades has resulted in increased development of national and regional infrastructures to reclaim effluents, which are mainly used for irrigation. Among reclamation techniques is the wetland method, which consists of pumping sewage water through several basins in which specific plants are grown to reduce the organic material load in the water. The reclaimed effluents at the exit of the wetlands contain lower amounts of organic material and microorganisms. However, the reclaimed water may be still infested with plant and human pathogens. Indeed, we observed the presence of coliform bacteria (in particular Escherichia coli) at high concentration in effluents from the Hod Hasharon wetland. Solar disinfestation was studied especially for soil (soil solarization) and found effective in controlling soilborne plant diseases. However, water solarization has not yet been studied for the control of human and plant pathogens. The objectives of the current study were to evaluate the feasibility of solar energy in disinfecting water in wetland systems. The research consisted of water ponds (100×100 cm) constructed of different depths. We examined water heating by capturing solar irradiation in the water under plastic covers. The tests were conducted year-round and included the control of bioassay bacteria (E. coli and Pectobacterium carotovorum subsp. atrosepticum). The solar irradiation during the summer months (June–September) resulted in elevation of water temperatures to values of 70, 60, 55, 50°C in the water containers at depths of 10, 20, 30, 40 cm, respectively. During the summer we obtained full control of the tested bacteria after 6 days. However, solar disinfection during the months of September–October yielded complete control of the bacteria only after 13 days. The application of solar disinfection in constructed wetlands can serve as a method for eliminating bacteria such as E. coli and P. carotovorum in the summer. This approach may enable safe use of this water for agricultural uses.

Survival of Clavibacter michiganensis in the soil and its influence on the disease in the following growth season

F. Abu Moch¹, R. Shulhani¹, M. Bornstein¹, M. Lufthouse², M. Sofer², D. Shtienberg¹ and O. Frenkel^1*

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: omerf@volcani.agri.gov.il]; and ² Negev R&D Center, M.P. Negev 85400, Israel

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is an important disease of tomatoes in the Negev region in Israel. The disease is initiated from a few plants that serve as the primary infection source within a growth structure, and spreads subsequently to the neighboring healthy plants. For many years it was assumed that the pathogen surviving in the soil and in root debris from one season serves as the main source of initial inoculum for the following season. However, in a set of observations carried out in commercial growth structures over several seasons, the correlation between disease intensity in two consecutive seasons was insignificant. The disease developed in only 28% of the examined commercial structures in the consecutive seasons. The infected structures were characterized by a large number of tomato fruits that remained on the ground. Several experiments showed that the pathogen is capable of surviving in tomato fruits and that fruit remains in the soil were at least as effective a source of initial inoculum as infested root debris. Seedlings planted 8 days after mixing the soil with Cmm-colonized fruits were significantly more diseased than seedlings planted in soil mixed with infected roots. Findings of our research suggest that tomato fruits may play an important role in bridging infection between the seasons and therefore should be removed efficiently.

Host preference of fungicide resistance in Pseudoperonospora cubensis

L. Falach^* and Y. Cohen

Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel [*e-mail: lidanf1@walla.com]

In a previous study we showed that in natural epidemics of cucurbit downy mildew the A1 mating type of the causal agent P. cubensis preferably attacks Cucumis species, whereas the A2 mating type prefers to attack Cucurbita species. In the present study, we grew nine cucurbits in a net-house (50–80 plants/species) during October–December 2013. Plants were inoculated with a mixture (1:1) of two single-sporangium isolates of P. cubensis: A1, resistant to mefenoxam (MFX), dimethomorph (DMM) and mandipropamid (MPD); and A2, sensitive to all three fungicides. Disease progress was monitored, and infected leaves were collected at 12, 19, 26 and 33 days post-inoculation (dpi) from each host and tested for the nature of the isolate harbored by each leaf. First disease symptoms appeared on cucumber and melon at 5 dpi, and at 11 dpi on Cucurbita moschata, squash, pumpkin and Lagenaria. Watermelon showed HR symptoms and Luffa was symptomless. At 33 dpi, percent infected leaf area on cucumber, melon, Beruti squash, C. moschata, Arlika squash, Lagenaria, pumpkin, watermelon and Luffa was 95%, 95%, 70%, 50%, 30%, 25%, 20%, 1% and 0%, respectively. A total of 120 isolates (one isolate/leaf) were collected during the epidemic. Percent isolates resistant to MFX in cucumber, melon, Lagenaria, C. moschata, Beruti squash, pumpkin, and Arlika squash was 100, 100, 93, 10, 0, 0 and 0%, respectively. Percent P. cubensis isolates resistant to MPD was 95%, 95%, 80%, 5%, 0%, 0%, and 0%, respectively, and to DMM: 80, 90, 80, 0, 0, 0 and 0%, respectively. Percent isolates showing the A1 mating type in these species was 95, 85, 18, 0, 0, 0, and 0%, respectively, and percent isolates showing the A2 mating type was 5, 10, 27, 100, 100, 100 and 100%, respectively. Percent isolates made of both A1 and A2 mating types was 0, 5, 55, 0, 0, 0 and 0%, respectively. Interestingly, no oospores were found in the sampled infected leaves. The data suggest that the A1 resistant isolate preferably attacks cucumber and melon, whereas the A2 sensitive isolate preferably attacks C. moschata, squash and pumpkin. Both isolates could attack Lagenaria.

Factors affecting the spread of Acidovorax citrulli in melon nurseries

M. Reuven¹, L. Chalupowicz¹, O. Dror¹, A. Koren², G. Kritzman¹, L. Mazor³, S. Burdman⁴ and S. Manulis-Sasson^1,*

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: shulam@volcani.agri.gov.il]; ² Hishtil Nurseries, Moshav Nehalim 49950; ³ Dept. of Agronomy and Natural Resources, ARO, The Volcani Center, Bet Dagan 50250; and ⁴ Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Bacterial fruit blotch (BFB) caused by the Gram-negative bacterium Acidovorax citrulli (Ac) is an economically important disease of cucurbits worldwide. Infested seeds are considered the most important source of primary inoculum for BFB. Because Ac is a quarantine pest in Israel, substantial efforts are taken to detect the pathogen in imported seeds. Many attempts have been made to develop effective means to control BFB. However, the disease is still considered a serious threat for the cucurbit industry, especially in the case of watermelon and melon crops. The objectives of this study were to: (a) examine the effect of irrigation methods and Kocide spray in reduction of the secondary spread of Ac; and (b) determine the role of different seedling organs in the secondary spread of Ac using a green fluorescence protein (GFP)-labeled strain. Experiments were conducted under nursery conditions in which foci of infested seeds were sown in the center of nursery trays. Overhead irrigation dispersed the pathogen from the primary inoculated seeds to 95% of the neighboring seedlings, with 80% of them displaying high disease severity. In contrast, sub-irrigation by floating did not lead to spread of the disease to the neighboring plants, which did not display disease symptoms or colonization by Ac. Kocide treatment after cotyledon emergence reduced disease incidence to 40%, with 37% of the plants displaying low disease severity. Determination of the presence of Ac in seedling organs emerging from infested seed demonstrated that the cotyledons were the most colonized. Ac was labeled by eGFP and used to study bacterial movement in melon seedlings. Confocal laser-scanning microscopy images of inoculated seedlings showed that the pathogen homogeneously colonized the lumen of xylem vessels and colonized mainly the cotyledons. Results of this study suggest that preventing the secondary spread of BFB in nurseries by sub-irrigation, in combination with cotyledon spraying with Kocide, may provide an effective means for disease control.

Effect of 'Superlon' application on Alternaria black spot disease of persimmon in the orchard

D. Ezra^* and D. Shtienberg

Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250, Israel [*e-mail: dezra@volcani.agri.gov.il]

Black spot disease of persimmon, caused by Alternaria alternata, affects fruit in the orchard and during storage. Since it was first described in Israel, attempts to control this disease in the orchard have been unsuccessful. Moreover, the incidence of diseased fruit in the orchards is steadily rising. In the orchard the symptoms are expressed as black lesions on the fruit skin or around the calyx, mostly in cracks. Fungicide applications in the orchard at different times during fruit growth did not reduce disease incidence when compared with untreated control plots. R. Stern, R. Ben-Arie & I. Ginzberg (2013) found that the application of 'Superlon' (cytokinin [6-benzyladenine] and gibberellins [GA4+7]) at early stages of fruit development reduced the incidence of calyx cracking in ‘Pink Lady’ apple, and as a result occurrence of Alternaria calyx rot was reduced. Calyx cracks are a major factor involved in Alternaria black spot of persimmon. We assumed that prevention of calyx cracks would reduce significantly disease frequency in the orchard. 'Superlon' was applied to a persimmon orchard at different times during fruit development and in different concentrations. Reduction in disease symptoms was observed in treated trees. Application of 0.2% 'Superlon' 70 and 100 days from leaf budding was the most effective treatment, with a 42% increase in asymptomatic fruits in the orchard, compared with the untreated control. It was also observed that asymptomatic treated fruits remained unaffected for a longer time in cold storage than asymptomatic untreated fruits from the control.

Prediction of resistance of Phytophthora infestans to mefenoxam using FTIR spectroscopy

A. Pomrantza^1,*, Y. Cohen², E. Shufan³, Y. Ben-Naim², A Salman³, S. Mordechai⁴ and M. Huleihel¹

¹ Dept. of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University, Be’er Sheva 84105 [*e-mail: amipo@bgu.ac.il]; ² Bar-Ilan University, Faculty of Life Sciences, Ramat Gan 52900; ³ Dept. of Physics, SCE-Sami Shamoon College of Engineering, Be’er Sheva 84100; and ⁴ Dept. of Physics, Ben-Gurion University, Be’er Sheva 84105, Israel

Phytophthora infestans, the causal agent of late blight, is a major pathogen of potato and tomato causing worldwide annual crop losses of billions of dollars. Fungicides controlling P. infestans have been used successfully for almost 100 years, the most common one in the last 30 years being the phenylamide fungicide mefenoxam. Since 1983 some P. infestans strains have developed resistance to mefenoxam. Identification and characterization of such resistant strains is important for disease control. Current methods, based on microbiological and molecular techniques, are expensive or time consuming. We suggest Fourier transform infrared spectroscopy (FTIR) combined with mathematical and statistical tools, as a method for characterization of resistant strains. FTIR, which is a reagent-free and inexpensive technique, can provide accurate results in only a few hours. In this study we applied FTIR to sporangia of P. infestans to examine the resistance to mefenoxam of field isolates. Our results show that FTIR spectroscopy enables classification of P. infestans isolates into mefenoxam-resistant and mefenoxam-susceptible with specificity of 83% and sensitivity of 81%.

BABA induces next-generation resistance against Bremia lactuca in lettuce

M. Vaknin^* and Y. Cohen

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel [*e-mail: moshe.vak@gmail.com]

BABA (β-aminobutyric acid) is known to induce systemic resistance against biotic and abiotic stresses in a large variety of plant species. Recently, it was reported that seeds collected from BABA-treated Arabidopsis plants produced plants resistant to Pseudomonas syringae pv. tomato DC3000 and Hyaloperonospora arabidopsidis. To examine such next-generation resistance, we sprayed BABA (S1) or water (C1) on lettuce, tomato and basil growing in net-houses during spring–summer 2013. The seeds were collected and their response to their respective pathogens – B. lactucae, Phytophthora infestans and Peronospora belbahrii – was examined. S1 lettuce seedlings at their cotyledon stage were more resistant (25%) against downy mildew compared with C1 seedlings. Challenge inoculation of lettuce at the 3–4-leaf stage resulted in similar disease development in S1 and C1 plants. S1 and C1 lettuce seedlings at the cotyledon stage were treated with various BABA concentrations (by spray or via the soil) and their response to B. lactucae was measured. The results showed that S1 developed resistance at lower concentrations of BABA as compared with C1 plants, suggesting intrinsic resistance of S1 plants. Similar experiments conducted with tomato and basil showed no difference in resistance between S1 and C1 plants. The data suggest that the next-generation resistance induced by BABA is host-dependent.

BACTERIAL DISEASES

Quorum sensing system of Xanthomonas campestris pv. pelargonii is involved in virulence and movement in geranium

L. Chalupowicz¹, V. Barel¹, M. Reuven¹, O. Dror¹, S. Burdman² and S. Manulis-Sasson^1,*

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: shulam@volcani.agri.gov.il]; and ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

The plant pathogenic bacterium Xanthomonas campestris (hortorum) pv. perlagonii (Xcp) is the causal agent of wilt and water-soaked lesions in geranium (Perlagonium hortorum), and is the most threatening bacterial disease of this ornamental in Israel and worldwide. Information about the pathogenicity mechanisms utilized by this pathogen and its interaction with the host plant is very limited. In a previous study we characterized the quorum sensing (QS) system and found that it is mediated by the diffusible signal factor (DSF). Mutagenesis of rpfF (encoding the DSF synthase) and rpfC (encoding the sensor kinase of the two-component system RfpC/RpfG) showed significant reduction in virulence of Xcp on geranium plants. The aims of the present study were to: (a) follow the movement and colonization patterns of Xcp in the host; and (b) measure virulence gene expression during early stages of infection. The wild-type strain and mutants in rpfF and rpfC were green fluorescence protein-labeled and used for inoculation of geranium plants. Confocal laser-scanning microscopy images of inoculated stems showed that the pathogen homogeneously colonized the lumen of xylem vessels and after 21 days reached the apex, colonizing the leaf mesophyll tissue intercellular spaces. In contrast, the mutants were confined to the inoculation site, forming a discontinuous bacterial growth of large aggregates which may occlude the vascular system and prevent movement. Biofilm formation in the presence of plant extracts showed that the mutants, but not the wild type, formed aggregates. Expression levels of putative virulence genes, virK and virB (encoding T4SS virulence factors), pelE1 (encoding a pectate lyase) and gumM (encoding an extracellular polysaccharide), were highly induced in the wild type during the 48 h following infection. In contrast, in rpfF and rpfC mutants no induction of these genes was detected. Results obtained indicate that QS is required for virulence, movement and colonization of Xcp in geranium.

Interaction between Verticillium dahliae and Dickeya solani in potato (Solanum tuberosum)

A. Siman Tov Vahaba^1,2, S. Burdman² and L. Tsror^1,*

¹ Dept. of Plant Pathology and Weed Research, ARO, Gilat Research Center 85280 [*email: tsror@agri.gov.il]; and ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Verticillium wilt, caused by Verticillium dahliae – a major disease of potato, can cause up to 50% yield loss. Slow wilt and blackleg of potato are diseases caused by the pectinolytic bacteria Dickeya solani and Pectobacterium spp. D. solani is introduced into Israel with seed tubers imported each year from Europe, causing serious economic damage. The object of the present study was to characterize the interaction between V. dahliae and D. solani in potato. In vitro studies showed a significant inhibition of V. dahliae development and mycelial growth when D. solani was present in the growth medium. In addition, increased formation of microsclerotia in the area adjacent to the bacterial colonies was observed. In planta DNA and colonization levels of V. dahliae were significantly lower in a co-inoculation treatment, compared with inoculation with the fungus alone. Confocal microscopy observations demonstrated reduction in development, germination of conidia and formation of new mycelium in the co-inoculation treatment. In in planta greenhouse studies high levels of D. solani in infected seed tubers reduced the rate and severity of infection of dry stems by V. dahliae, and decreased the incidence of symptomatic V. dahliae-infected plants and low levels of V. dahliae in the progeny tubers, compared with seed tubers bearing low levels of D. solani. The results of the present study suggest an antagonistic relationship between V. dahliae and D. solani in potato, both in vitro and in planta.

Involvement of type IV pilus and phenotypic variation in virulence of Acidovorax citrulli

T. Rosenberg*, R. Shrestha, D. Makarovsky and S. Burdman

Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel [*e-mail: tally.rosenberg@mail.huji.ac.il]

Acidovorax citrulli (Ac) causes seedling blight and bacterial fruit blotch of cucurbits. Serious economic losses caused by Ac in cucurbits have been reported in many parts of the world. Because of the highly destructive character of the pathogen, the lack of efficient disease control methods and an absence of resistance sources, bacterial fruit blotch represents a serious threat to the cucurbit industry worldwide. To identify genes contributing to Ac virulence, our group generated a library of transposon mutants and screened it by virulence assays, leading to the discovery of several mutants with reduced virulence. Many of the isolated mutants were found to be impaired in genes encoding for biosynthesis of type IV pili (TFP). The goal of this research is to discover more TFP-related genes and to determine the role played by TFP in Ac virulence. In the course of routine laboratory work Ac colonies with different morphology relative to the wild type were detected. These are phenotypic variants (PV) of the bacterium. The PVs do not produce typical TFP-mediated twitching motility haloes around the colony, and transmission electron microscopy revealed that, indeed, the PVs lack TFP. Additionally, as similar as TFP mutants, the tested PVs possess reduced virulence relative to the wild type. We are currently characterizing morphological, physiological and genetic alteration in the PVs to characterize the phenomenon of phenotypic variation in Ac.

The recovery phenomenon as a potential tool to decrease phytoplasma yellows disease in grapevine

V. Naor^1,2,*, T. Zahavi³ , R. Bordolay¹, R. Berkowitz¹, A. Wassermann², M. Harel², E. Gol², and M. Dafny-Yelin¹

¹ Golan Research Institute – Haifa University, Qazrin 12900 [*e-mail: vered_n@macam.ac.il]; ² Ohalo College, Qazrin 12900; and ³ Extension Service, Ministry of Agriculture and Rural Development, Qiryat Shemona 10200, Israel

Spontaneous recovery is a unique phenomenon in vines infected with yellows disease (YD). Recovery occurs in the year following infection by phytoplasma. Recovered vines regain normal growth within another 2–3 years and are considered less sensitive to re-infection. Assuming this trait results from stimulation of plant resistant mechanisms, it was not clear whether recovered vines can serve as propagation material of ‘resistant’ plants. Our objectives were to compare growth parameters and sensitivity to re-infection in plants propagated from recovered vines (Rec) and healthy vines (H). Three hundred plants (half rooted and half grafted on the scion ‘Richter 110’) were planted in pots in a net-house and in an open field. In the net-house the plants were subjected to controlled infection by placing specimens of the insect vector on each plant or by bud grafting. In the open vineyard the plants were subjected to natural infection. Growth parameters were monitored in these plants for 3 years and in plantlets in tissue culture. Growth of Rec plants did not differ from that of H plants. In the second year, pruning weight of plants in the net-house was similar to plants in the field, but grafted plants were bigger than rooted plants. The difficulty of controlled infection and the long period required for symptom appearance prevented testing for sensitivity to re-infection. However, symptoms of YD were observed in 11/50 grafted plants regardless of vine source (H or Rec). We will continue to monitor YD symptoms in the tested vines to address this question.

GENETICS, RESISTANCE AND RESISTANCE INDUCTION

Resistance of tomato lines to bacterial canker caused by Clavibacter michiganensis subsp. michiganensis

Y. Rekah*

The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel [*e-mail: yael.rekah@mail.huji.ac.il]

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis causes severe damage to tomatoes worldwide. Few sources of resistance have been reported. We screened for resistance sources originating from Solanum lycopersicum. Two open-pollinated lines, Cocabul and line NK712, were found to be highly resistant to the disease in temperature-controlled greenhouse tests, as well as under field conditions. Crosses that were carried out between these two sources and a highly susceptible line, Motelle, suggest that the resistance is apparently controlled by a single major gene with a dominant effect. Evaluations of the crosses between Cocabul and Motelle revealed that 95.5% and 88.2% of the F1 plants were resistant in greenhouse tests and in field experiments, respectively. In the same tests performed on the F1 plants of the cross between NK712 and Motelle, 99% of the plants were resistant in the greenhouse tests and 100% in field experiments. Segregation of an F2 population of Cocabul and Motelle crosses showed 81.3% and 82.3% plants resistant in the greenhouse and field experiments, respectively. Segregation of F2 population of NK712 and Motelle cross was 73.4% and 76.4% resistant in the greenhouse and field experiments, respectively. The populations of F1 and F2 crosses of both resistance sources and Motelle corresponded to the resistant/susceptible ratios expected for a major dominant gene inheritance. However, in some the tests carried out for segregating populations of the BC1 of the two resistance sources, the expected ratio of 50% resistant/susceptible plants was not obtained in all cases. Our studies showed a good correlation between the results obtained in the greenhouse tests which we developed, and those obtained under field conditions.

First identification of Fusarium oxysporum f.sp. lactucae as the causal agent of lettuce wilt disease and breeding of resistant lettuce varieties

Y. Rotem^* and A. Nir

Hazera Genetics Ltd., Mivhor M.P., Lakhish Darom 79354, Israel [*e-mail: yanivr@hazera.com]

Fusarium wilt of lettuce, which is caused by Fusarium oxysporum f.sp. lactucae, was discovered for the first time in Japan in 1955, and later identified in other countries. Three races (1, 2 and 3) of this pathogen are known. A severe new wilt disease of lettuce appeared in 2009 in the Besor region and other locations in Israel. We have isolated F. oxysporum f.sp. lactucae from diseased lettuce plants in Sede Nizzan (Besor region), and established that it is the causal agent (by Koch's postulates). We have identified our isolate as race 1 of lettuce Fusarium by inoculation of a known differential set. Hundreds of lettuce breeding lines and varieties were screened for Fusarium resistance in laboratory tests, and a few lines (both ‘Romaine’ and ‘Iceberg’ types) were found to be highly resistant to Fusarium. Many field observations were made in a heavily infested field on Asaf Farchi's farm, Moshav Sede Nizzan. There is a good correlation between laboratory results and field observations. Following these results, a first commercial Fusarium-resistant lettuce variety was released – the variety ‘Asaf’.

Characterization of plant defense induction by the epiphytic fungus Pseudozyma aphidis

S. Alster^*, A. Dafa-Berger, A. Gafni, O. Barda, K. Buxdorf and M. Levy

Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel [*e-mail: shanee.alster@mail.huji.ac.il]

Plant pathogens challenge our efforts to maximize crop production due to their ability to develop resistance to pesticides. Fungal biocontrol agents have become an important alternative to chemical fungicides due to environmental concerns regarding conventional pesticides. We isolated the epiphytic yeast-like fungus Pseudozyma aphidis. Our findings demonstrate that the isolate secretes extracellular metabolites that inhibit plant pathogens in vitro, and that the population of P. aphidis on the surface of a healthy plant increases in the presence of pathogens. It was found that P. aphidis suppresses callose deposition, part of the plant basal immune response activated by microbe-associated molecular pattern (MAMP) recognition. Moreover, P. aphidis suppresses the callose deposition that is elicited by other MAMPs, like Flg22 and chitin. P. aphidis can trigger MAMP-activated reporter genes, such as MYB51, WRKY11 and At5g25260. P. aphidis also triggers a strong response in Arabidopsis leaves, like other MAMPs (Flg22 and chitin, for example). In work with Arabidopsis mutants in the plant hormonal defense we found that MYC2 plays a major role in this MAMP response. MYC2 is a transcription factor known to be involved in the priming for enhanced induced systemic resistance associated with beneficial microbes. In conclusion, P. aphidis suppresses MAMP-elicited callose deposition to enable its establishment on the plant surface, but also primes the plant's systemic defense response to act against pathogens.

Improving resistance against late blight in tomato by pyramiding resistance genes from Solanum pimpinellifolium and Solanum habrochaites

Y. Ben Naim^* and Y. Cohen

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel [*e-mail: Yar2710@gmail.com]

Late blight caused by the oomycete Phytophthora infestans is a major disease of tomato (Solanum esculentum) and potato (Solanum tuberosum). The genes Ph-1, Ph-2 and Ph-3, derived from the wild tomato species Solanum pimpinellifolium, were incorporated in recent decades into a number of tomato breeding lines to achieve resistance against late blight. However, these genes are not effective in certain countries, including Israel. A scan conducted with 200 isolates of P. infestans revealed that Ph-1, Ph-2 and Ph-3 were fully effective against 1%, 40% and 70% of the isolates, respectively. Therefore, we combined the last two genes into a single elite tomato line. F4 plants carrying both Ph-2 ^+/+and Ph-3 ^+/+showed a spectrum of resistance expanded by 15–40%, and the disease level was reduced by 30–100% relative to plants bearing the two genes separately. However, although the lines carrying Ph-2 ^+/+/Ph-3 ^+/+genes were more resistant and had a wider resistance spectrum, they were still susceptible to ~20% of the isolates. The wild type tomato Solanum habrochaites line LA1033 was found to be resistant to 98% of the 200 isolates. To introduce the resistance of LA1033 into Ph-2/Ph-3 lines, we first crossed LA1033with a susceptible tomato line. BC-4 lines carrying the two resistance segments (QTL's) Lb'4 and Lb'11a, derived from LA1033, were highly resistant against isolates that attack plants carrying Ph-2, Ph-3, or both. Next, we shall incorporate the resistance from LA1033 into the lines carrying Ph-2 and Ph-3.

Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum

Y. Meller Harel^1,3*, Z.M. Haile^2,3, N. Okon^3,4, Y. Elad³, E. Rav-David³, M. Borenshtein³, E. Jurkevitch⁴ and J. Katan⁴

¹ Plant Protection and Inspection Services, Ministry of Agriculture and Rural Development, Bet Dagan 50250, Israel [*e-mail: yaelm@moag.gov.il]; ² Istituto Agrario di San Michele all’Adige (IASMA), 38010 Trento, Italy; ³ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250, Israel; and ⁴ Dept.of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

The effect of soil solarization (SH) and soil treatment with Trichoderma harzianum (TH) isolate T39 on induced resistance to grey mold (GM) – caused by Botrytis cinerea, and powdery mildew (PM) – caused by Podosphaera xanthii, was studied. Plants grown in soils pretreated with the two agents (SH and TH), alone or in combination, were later submitted to leaf inoculation, thus separating the control agents from the pathogen. There was a significant reduction in incidence or severity of GM in pretreated cucumber, strawberry, bean and tomato plants and of PM in cucumber, with a greater decrease when SH and TH were combined. The general bacterial community (and more specifically bacilli, pseudomonads and actinobacterial communities) of the strawberry rhizosphere were affected by the treatments, as revealed by denaturing gradient gel electrophoresis. Expression levels of defense-related genes were tested in tomato plants following soil treatment by SH and TH. The soil treatments affected the expression of salicylic acid (SA)-, ethylene (ET)-, and jasmonic acid (JA)-responsive plant genes. In both cases, genes related to SA and ET – PR1a, GluB, CHI9, and Erf1 – were down-regulated, while additionally the JA marker gene PI2 was up-regulated by SH and TH. The up-regulation of the above genes upon B. cinerea infection reveals a priming effect of both soil treatments. The present study shows that, in addition to their known suppression activity on the pathogen, SH and TH have the capacity to induce resistance in various plants. It also suggests that induced resistance by SH is partly due to increased rhizobacteria populations.

DISEASE CONTROL – NEMATODES

Monitoring the root-knot nematode in soil samples by using quantitative real time PCR approach

D. Shtriker^1,2*, I. Glazer¹, A. Gamliel³ and S. Brown Horwitz¹

¹ Dept. of Entomology, Nematology and Chemistry Units, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: daniel.shtriker@mail.huji.ac.il]; ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100; and ³ Laboratory for Pest Management Research, Institute of Agricultural Engineering, ARO, The Volcani Center, Bet Dagan 50250, Israel

Meloidogyne javanica is considered to be one of the most destructive nematodes worldwide. This nematode damages a variety of plants, including ornamentals, flowers, vegetables and other crops. In Israel, peppers as well as tomatoes are the main crops damaged, reducing yield by up to 30%. Currently, monitoring the nematode before planting is uncommon, for two main reasons: (a) the high cost of monitoring soil samples, and (b) the need for many samples for appropriate analysis and field coverage. Furthermore, the number of taxonomy specialists in nematology is decreasing and such expertise is becoming rare. Nevertheless, a reliable and rapid technique is needed to replace the traditional identification method. Quantitative Real Time PCR (qRT-PCR) has been used for qualitative and quantitative identification of M. javanica. First, a suitable DNA purification protocol, with minimal DNA loss, has been determined. Using specific primers to the genus Meloidogyne, a standard curve was built and has been found to be reliable in the identification of a known number of nematodes, without the need for taxonomic expertise. Using that tool with the appropriate primers for each Meloidogyne species we will be able to identify those species in different soils, especially at the J₂ stage. Information that will be accumulated from the current research will be important for monitoring the nematode following different soil treatments and measuring the efficiency of integrated nematode management that will be developed.

Gene networks and lipid signals governing plant interactions with the root-knot nematode Meloidogyne javanica

I. Iberkleid^1,2*, N. Sela¹ and S. Brown Horowitz¹

¹ Dept. of Entomology, Nematology and Chemistry Units, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: ionit.iberkleid@mail.huji.ac.il]; and ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Plant parasitic nematodes produce a unique class of fatty acid and retinol-binding proteins (FAR) with no counterpart in their hosts. Meloidogyne javanica’s fatty acid and retinol-binding protein (Mj-FAR-1) is critical for juvenile and sedentary development, as well as giant cell development, presumably by modulating plant lipid signals and defense. For further insights into the role of Mj-FAR-1 in disease development, we analyzed RNA-seq profiles of tomato hairy roots where mj-far-1 is constitutively expressed, compared with control roots, at early and late stages after inoculation. RNA-seq revealed that 3970 transcripts were differentially expressed: 2069 up-regulated and 2205 down-regulated in the comparison of mj-far-1 overexpressing line with control root line, when summing all inoculated and non-inoculated samples. A total of 61 differentially expressed genes overlapped between all transgene overexpressing root samples. This gene list might provide initial understanding of the mj-far-1 associated increases in susceptibility observed in the overexpressing root line. These genes include fatty acid metabolism, hormone signaling (jasmonic acid and auxin-related), biotic stress-related genes and the phenylpropanoid pathway. Validation of the data was confirmed with quantitative real-time PCR of representative genes and through the use of a β-glucuronidase reporter gene downstream of candidate gene promoters. Overall, the RNA-seq analysis indicates that, at the early time points, samples clustered predominantly in relation to the differential genetics, while at later time points samples clustered in relation to temporal dynamics of the nematode infection. Our results will uncover the role of lipid signals and mj-far-1 in defining a common transcriptome that facilitates nematode infection.

New nematological problems in greenhouse vegetables (pepper, tomato(

E. Kozodoy^* and E. Gomberg

Plant Protection Diagnostic Service, Plant Protection and Inspection Services (PPIS), Ministry of Agriculture and Rural Development, Bet Dagan 50250, Israel [*e-mail: evgenik@moag.gov.il]

Nematological problems in vegetable greenhouse cause heavy crop damage. The most common nematodes are well known to growers and belong to different species of root-knot nematodes. They can lead to dramatic damage, but diagnosing the symptoms is relatively easy, and in most cases does not require laboratory testing. At medium and higher infection rates, many root galls can be observed. In recent years new damage has been observed in vegetable greenhouses caused by lesion nematodes (Pratylenchus penetrans) and needle nematodes (Longidorus elongatus), which are more difficult to diagnose. Root-knot nematode-infected roots present black lesions, are necrotic, thin and break easily. Plant development is slow and fruits are smaller. These symptoms are similar to those caused by fungal and bacterial diseases. Plants infected by needle nematodes are short with chlorotic leaves and their roots are thickened and short. Diagnosis requires laboratory testing. These nematodes have to be taken into account together with root-knot nematodes when looking for resistant plants.

VIRUSES AND VIRAL DISEASES

Symptom appearance of Grapevine leafroll disease and its effect on vine physiology and grape quality in GLRaV-3-infected vines in northern Israel

R. Nilovisky^1*, O. Crane ¹, G. Sapir¹ and T. Zahavi²

¹ Northern R&D, Qiryat Shemona 11016; and ² Ministry of Agriculture and Rural Development, Extension Service, Northern Region, Israel [*e-mail: rotkenel@gmail.com]

Nine or ten viruses of the Closterovirideae are associated with Grapevine leafroll disease and are therefore named Grapevine leafroll-associated viruses (GLRaV1-10). In Israel, GLRaV3 is abundant. The effects of the disease are well documented, although there are differences in symptom severity, both between vineyard plots and between vines in the same plot. In a survey conducted in three vineyards, a good correlation was found between number of days before harvest at which disease symptoms are first observed, and the severity of the symptoms at harvest. The correlation slope was affected by the year (P=0.061) more than by the vineyard plot (P=0.3), suggesting an effect of the weather during the growing season. Five to ten percent of the symptomatic vines in a previous year did not display any symptoms in the following year. Brix level of vines with low disease severity (23.9%) did not differ significantly from non-symptomatic vines (25.1%), but both differed significantly from high symptom severity vines (21.9%). Berry color was the most affected. Yields of symptomatic vines tended to be lower than those of non-symptomatic ones, but berries were significantly bigger in most symptomatic vines. Real Time PCR analysis showed that some of the non-symptomatic vines were also infected and there was no significant difference in the virus titer between vines with different disease severity. When comparing infected symptomatic (1-1) vines to infected-not-symptomatic (1-0) and to healthy vines (0-0), we found that Brix level, berry size and water status of 1-0 vines were closer to those of 0-0 than to 1-1 vines.

Nuclear actin filaments in plant virus infection sites

A. Levy^*, J. Zheng and S. G. Lazarowitz

Dept. of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14850, USA [^* e-mail: al658@cornell.edu]

Viruses rely on host functions for their replication and spread, and must therefore hijack, inhibit or redirect cellular functions while avoiding host defenses. Successful viral infections result from integration of protein–protein interactions with changes in host gene expression. Plant viruses also need to overcome the barrier of the cell wall by encoding movement proteins (MPs). The exemplar of how a single MP regulates and coordinates these activities is the Tobamovirus Tobacco mosaic virus (TMV) MP. TMV does not infect the model plant Arabidopsis thaliana, but Turnip vein-clearing virus (TVCV) is a related Tobamovirus which does infect this host, and can serve as a model to take advantage of Arabidopsis genomic resources to study virus movement. However, TVCV findings have been interpreted based on the unproven premise that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed an MP^TVCV-GFP fusion and report the unexpected discovery that MP^TVCV, in addition to localizing to ER membrane and plasmodesmata, targets the nucleus and induces the formation of F-actin-containing filaments that associate with chromatin. Mutational analyses showed that nuclear localization of MP^TVCV was necessary for TVCV local and systemic infection in both Nicotiana benthamiana and Arabidopsis, but had no effect on MP^TVCV cell-to-cell movement, its binding to nucleic acids, or viral replication. These studies identify a novel nuclear stage in TVCV infection and suggest that, in the nucleus, MP^TVCV associates with nuclear actin in chromatin remodeling or modifying complexes to reprogram transcription of host defense genes.

Immunity to Tomato yellow leaf curl virus in transgenic tomato is associated with transgene small RNA accumulation

D. Leibman^1,*, D. Wolf², P. Shanmugam², S. Haviv¹, M. Lapidot² and A. Gal-On¹

¹ Dept. of Plant Pathology and Weed Research, and ² Dept. of Vegetable Research, ARO, The Volcani Center, Bet Dagan 50250, Israel [*e-mail: diana@agri.gov.il]

Tomato yellow leaf curl virus (TYLCV) has become a major limiting factor in tomato production both in Israel and many other regions. Classical crop breeding for disease resistance in tomato is efficient against TYLCV and based on a number of resistance genes; however, transmission of virus resistance to other species is very difficult. In contrast to classical breeding, transgenic resistance provides immunity based on a dominant gene and possibly can be utilized easily in other species. Post-transcriptional gene silencing (PTGS) is a natural defense response of plants against invading viruses. Here we applied this approach against the DNA virus TYLCV in its natural host – tomato. Tomato plants were transformed by Agrobacterium with a hairpin silencing construct expressing transgene-double-strand-RNA (tr-dsRNA). The construct contains three consecutive partial fragments of the IR, V2 and C2 viral genes under control of a constitutive viral promoter. Fifty independent transgenic lines showed different levels of resistance. Nine lines which showed ‘immunity’ were used for further analyses, including one marker-free line (without NPTII gene). Selected transgenic lines expressed transgene-small-interfering RNA (tr-siRNA) of 21–24 bases. The homozygotic progeny of independent lines displayed high levels of resistance to different TYLCV strains by whitefly infection. Deep sequencing analysis of the transgenic and grafted tomato plants was performed to study tr-siRNAs distribution and movement. The grafting experiment did not display resistance in non-transgenic scions, although movement of tr-siRNA from transgenic rootstock to non-transgenic scion was observed.

Investigating interactions between the whitefly Bemisia tabaci, the bacterial endosymbiont Rickettsia, and Tomato yellow leaf curl virus

A. Kliot^1,2,*, H. Czosnek² and M. Ghanim¹

¹ Dept. of Entomology, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: adi.kliot@mail.huji.ac.il]; and ² The Robert H. Smith Institute of Plant Sciences and Genetics, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

The whitefly Bemsia tabaci is a cosmopolitan polyphagous insect pest that transmits over 100 plant viruses, of which Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: begomovirus) is devastating to tomato crops worldwide. TYLCV is transmitted exclusively by B. tabaci in a persistent circulative manner. In this mode of transmission, the virus is ingested with phloem, crosses the midgut to enter the hemolymph, and reaches the salivary glands from which it is transmitted during whitefly feeding. Previous research in our laboratory had shown that a GroEL protein produced by the insect secondary endosymbiont Hamiltonella is implicated in the transmission of TYLCV. The protein interacts with the virus and is assumed to protect virions from proteolysis in the hemolymph. Our recent results indicate that Rickettsia, another secondary endosymbiont of B. tabaci, may influence TYLCV–B. tabaci interactions. Unlike other secondary endosymbionts in B. tabaci, which reside in a specialized organ, Rickettsia occupies most of the body cavity of the insect, including organs implicated in virus transmission. TYLCV transmission by B. tabaci Rickettsia-infected and uninfected iso-female strains show a greater ability of the Rickettsia-infected population to transmit the virus. Furthermore, virus levels within Rickettsia-infected whiteflies were significantly higher than those in the Rickettsia-uninfected ones. We further found indications for an antagonistic relationship between Rickettsia and TYLCV: Rickettsia levels decline significantly upon virus acquisition and rise only after virus titers drop. Future research will focus on investigating TYLCV–Rickettsia interactions within B. tabaci and the implications of these interactions on virus retention and transmission.

Potato virus Y Real Time PCR signal is differentially maintained in aphids following loss of virus transmission ability

S. Prakash¹, N. Luria¹, Y. Tam¹, A. Dombrovsky¹, Z. Dar² and V. Gaba^1,*

¹ Dept. of Plant Pathology and Weed Science, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail vpgaba@volcani.agri.gov.il]; and ² Extension Service, Ministry of Agriculture and Rural Development, Bet Dagan 50250, Israel

Potyviruses are transmitted to host plants in a non-circulative manner by a wide range of aphid vectors. A Real Time Polymerase Chain Reaction technique was developed to detect Potato virus Y (PVY), an important potyviral pathogen of solanaceous crops. With this technique PVY could be detected in dormant potato (Solanum tuberosum) tubers and in individual aphids growing on greenhouse-grown PVY-infected pepper (Capsicum annuum ) plants. The aphid Real Time PCR signal was confirmed by Northern blot. The acquisition and retention of PVY by two important aphid pest species, Myzus persicae Sulzer (green peach aphid) and Aphis gossypii Glover, were examined by Real Time PCR and compared with virus transmission to pepper plants. Measured by Real Time PCR, 100% of individual M. persicae acquired PVY and retained the virus signal during a 4-h starvation period. However, only 80% of A. gossypii acquired PVY, and retention of the virus signal declined to 50% of individual insects after about 75 min starvation. Transmission to pepper plants by groups of aphids (50 per plant) was tested: 100% infection was observed for both aphid species with no starvation period. Starvation for 1 h or more resulted in zero transmission for both aphid species. A. gossypii were observed in a field plot with PVY-infected potato plants. Aphids bearing a Real Time PCR signal were found on both infected and uninfected potato plants. PVY-signal-bearing aphids were detected alongside non-PVY carriers on both infected and uninfected potato plants.

EMERGING DISEASES

Fusarium crown rot in cucumber and melon: studying the biology of the disease

G. Orgil^1,2,*, R. Cohen¹ and O. Yarden²

¹ Unit of Cucurbit Research and Breeding, Newe Ya'ar Research Center, ARO, Ramat Yishay 30095 [*e-mail: gidi.orgil@gmail.com]; and ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Crown rot disease in cucumber and melon, caused by the pathogen Fusarium oxysporum radicis cucumerinum (F.o.r.c), was first identified in Israel in 2000. Recently, it also attacked melons in different areas. This disease differs from Fusarium wilt by the appearance of external rotting and air dispersal of the spores. Presently, the disease is controlled by applying fungicides to the soil and grafting cucumber and melons onto resistant rootstocks. Information regarding pathogenesis and resistance mechanisms is scarce, and there are many questions regarding the host–pathogen interaction. Therefore, the goals we set for the study are: (i) studying the pathogen's host range; (ii) studying the host–pathogen interaction, focusing on the colonization process and involvement of mycotoxins; and (iii) evaluating the potential of grafted plants for disease control, focusing on resistance induction in a susceptible scion by a resistant rootstock. It was found that the pathogen is able to penetrate the host through the root and through a wound in the stem. This trait enables us to inoculate a susceptible plant grafted on resistant rootstocks through the scion's stem. It was also found that resistant melon rootstocks induce a partial resistance in the scion. Microscopic examination revealed that when colonizing a susceptible plant, the fungus propagates through the intercellular spaces to the xylem, through which it spreads systemically. In a resistant plant, however, penetration to the xylem is hindered. Our findings provide new knowledge of the pathogenesis and resistance mechanisms that can be used for developing control strategies and rational breeding for resistance.

Management of Fusarium proliferatum in onion

Y. Isack^1,2,*, M. Benichis², J. Gatker², D. Gillette³ and A. Gamliel²

¹ Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100 [*email: yochai.isack@gmail.com]; ² Institute of Agricultural Engineering, ARO, The Volcani Center, Bet Dagan 50250; and ³ Ardom R&D, Southern Arava, Israel

Fusarium proliferatum is a soilborne pathogen which infects important agricultural crops such as maize, onion and garlic. The symptoms on white onion bulbs appear as a salmon-colored blotch on external scales. However, the fungus does not generate external symptoms in other onion cultivars (yellow and red) or other hosts. Infected seeds and onion sets are an important means for pathogen distribution to new fields. F. proliferatum can also cause bulb rot in the field or in storage, and is able to produce several mycotoxins, such as Fumonisin B₁, in infected tissues. The objectives of the current study were to develop measures to manage F. proliferatum and reduce infection during onion production. Dipping of onion sets in a suspension of systemic fungicides, soil treatments, drench application of systemic fungicides during the season and combinations of these treatments were tested. Pretreatment with systemic fungicides were not effective in reducing populations of F. proliferatum in the tissues of onion sets. Soil fumigation with either metam sodium or dimethyl disulfide can decrease fungus soil inocula density. A combination of the fumigants with soil solarization further improves pathogen control. Drench application with benomyl at the rate of 20 g l ^-1 provided effective control of F. proliferatum in pot experiments, but application of the fungicide in the field provided only partial control of the pathogen. These findings emphasize the limitation of fungicides in controlling the pathogen in infected tissues. Hence, the most important component in ensuring a healthy crop is providing pathogen-free propagation material.

Rosellinia necatrix in deciduous orchards: Evaluation of pathogen distribution

M. Dafny-Yelin^1,*, R. Bordelei¹, S. Nasralla², A.M. Safadi³, P. Safadi⁴, S. Freeman⁵, M. Taiseer², S. Kfir⁶, O. Levi⁶ and M. Miron⁶

¹ Golan Research Institute, Haifa University, Qazrin 12900 [*e-mail: merydy@gri.org.il]; ² Elmagareq Agricultural Cooperative, Majdel Shams 12438; ³ El-Sharaq Agricultural Cooperative, Majdel Shams 12438; ⁴ Saar – Agricultural Cooperative, Mas`ade 12435; ⁵ Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250; and ⁶ MIGAL-Northern Research & Development, Qiryat Shemona 11016, Israel

White root rot disease caused by Rosellinia necatrix (anamorph Dematophora necatrix) is destructive to many fruit trees, particularly apple and other deciduous crops. In the north of Israel farmers have reported widespread wilting of deciduous trees, but the origin and true impact of this problem is poorly understood. R. necatrix and other soilborne pathogens were often thought to be the causal pathogen. Today there is no treatment for R. necatrix infection in Israel, and the disease spreads to new trees each year. The research objective was to evaluate R. necatrix damage in orchards in the northern region. During the study ca. 130 root samples were collected from diseased orchards and analyzed for the appearance of typical mycelia of the pathogen. R. necatrix root infection was found in ca. 40 plots. In five locations, most of the infected plots were adjacent to Mediterranean forests, whereas in three others the soil was not always native. In many cases, farmers have more than one infected plot, probably as a result of soil transfer from one plot to another, via infected tools. The results may help raise awareness and prevent infection by increasing the distances from Mediterranean forests and minimizing R. necatrix spread from infected plots.

Interceptions of fungal plant diseases in imported apples and pears

E. Levy*, G. Elkind, E. Teverovsky, R. Gofman, Y. Meller Harel, E. Gomberg and I.S. Ben-Ze’ev

Plant Protection Diagnostic Service, Plant Protection and Inspection Services (PPIS), Ministry of Agriculture and Rural Development, Bet Dagan 50250, Israel [*e-mail: ednalg@moag.gov.il]

The PPIS Diagnostic Service laboratories routinely test imported fruits, vegetables and propagation material for pests and pathogens (bacterial, fungal and viral). A few years ago (1998, 2000, 2005) we reported interceptions of Phacidiopycnis pyri (Fuckel) Weindlymayr, a causal agent of stem-end rot of apples and pears. Since Phacidiopycnis species are not present in Israel, they are regarded as quarantine pests. A new stem-end disease of apples was found in Washington State in 2005 and a new species, Phacidiopycnis washingtonensis, was described. Last year infected apple fruits with similar symptoms imported from the USA were intercepted. Since the distinction between P. pyri, P. malorum and P. washingtonensis requires comparison of several DNA sequences, we identified the recently intercepted pathogen just as Phacidiopycnis sp. until the molecular results become available. In addition, Neofabraea malicorticis H.S. Jacks, the causal agent of bull's eye rot, was identified from infected apples imported from the USA. The symptoms include circular brown rot, yellowish in the center with a darker perimeter. Diagnosis was based on the shape, size and color of the acervuli, conidia and DNA sequence data. In orchards, the fungus enters the trees through pruning wounds and due to stress conditions like frost cracks. At harvest the fungi of these two diseases are in a latent condition in the fruit and the symptoms become visible in storage or after maritime shipment.

PHYSIOLOGY AND RESISTANCE

Divergence of Acidovorax citrulli into three lineages based on type III secreted effector sequence analysis

N. Levi^1,*, T. Zimermann¹, M. Živanović ², S. Traore³, C. Spröer⁴, B. Zhao³, G. Welbaum³, R. Walcott², J. Sikorski⁴ and S. Burdman¹

¹ Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel [*e-mail: noam.levi2@mail.huji.ac.il]; ² Dept. of Plant Pathology, The University of Georgia, Athens, GA 30602, USA; ³ Dept. of Horticulture, Virginia Polytechnic Institute, Blacksburg, VA 24061, USA; and ⁴ Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany

Bacterial fruit blotch (BFB) is a threatening disease of cucurbits, caused by the Gram-negative bacterium Acidovorax citrulli. The disease gained importance after devastating outbreaks in watermelon fields in the USA during the 1980's. Recently, serious economic losses due to BFB have been reported for other cucurbit species in many parts of the world. Today, there are no effective means to cope with BFB, which represents a serious threat to the cucurbit industry, mainly watermelon and melon. Despite the economic importance of BFB, relatively little is known about basic aspects of the disease. According to host range, and biochemical and genetic features, at least two groups exist within this species: group I includes strains that were isolated mainly from non-watermelon hosts, while group II includes watermelon strains. As with many Gram-negative plant pathogenic bacteria, A. citrulli requires a type III secretion system (T3SS) for pathogenicity. The T3SS injects effector proteins directly into the cytosol of the plant cells, which allow the pathogen to manipulate the host cellular activities to its own benefit. Due to the important role of these effectors in pathogenicity, we hypothesized that the observed group host preferential association is, at least partially, influenced by their effector repertoire. In this study we cloned and sequenced the effector genes from 22 A. citrulli strains isolated from different host plants and geographic locations. Comparative analyses of the effector genes revealed that these genes cluster according to the group I/II classification. Moreover, our analyses, combined with additional experimental evidence, led to the identification of a third group of A. citrulli strains. Currently, we are investigating selected effectors to assess their contribution to virulence and host preferential association.

The mycotoxin patulin contributes to pathogenicity of Penicillium expansum

S. Barad ^1,*, S. Brown Horowitz², A. Sherman³ and D. Prusky¹

¹ Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250 [^* e-mail: barad_shiri@yahoo.com]; ² Nematology Unit, Institute of Plant Protection, ARO, The Volcani Center, Bet Dagan 50250; and ³ Dept. of Genomics, ARO, The Volcani Center, Bet Dagan 50250, Israel

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest fruit maceration through secretion of D-gluconic acid (GLA) and secondary metabolites such as the mycotoxin patulin in colonized tissue. While GLA involvement in pathogenicity has been suggested, the mechanism of patulin accumulation and its contribution to P. expansum pathogenicity remain unclear. The roles of GLA and patulin accumulation in P. expansum pathogenicity were studied using: (i) GOX2-RNAi mutants exhibiting decreased GOX2 expression, GLA accumulation and reduced pathogenicity; (ii) IDH-RNAi mutants exhibiting down regulation of IDH (the last gene in the patulin biosynthesis pathway), reduced patulin accumulation and no effect on GLA level; and (iii) PACC-RNAi mutants exhibiting down-regulation of both GOX2 and IDH, that decreased GLA and patulin production. Present results indicate that conditions enhancing the decrease in GLA accumulation by GOX2-RNAi and PACC-RNAi mutants, and not low pH, affected patulin accumulation, suggesting GLA production as the driving force for further patulin accumulation. Thus, it is suggested that GLA accumulation may modulate patulin synthesis as a direct precursor under dynamic pH conditions modulating the activation of the transcription factor PACC and the consequent pathogenicity factors, which contribute to host-tissue colonization by P. expansum.

Involvement of the NADPH oxidase complex in development and pathogenicity of Colletotrichum acutatum on strawberry

T. Gershon^1,2,*, S. Freeman¹ and O. Yarden²

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: tomer_gershon1@walla.com]; and ² Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Colletotrichum acutatum J.H. Simmonds (C.a) is a major causal agent of strawberry anthracnose. Reactive oxygen species (ROS) generated by NADPH oxidase (Nox) play a key role in fungal growth, development and pathogenicity. We characterized four components of the C.a Nox system encoded by the genes nox1, nox2, rac1 and noxR, and studied the function of three of these genes using a reverse genetic approach. Δnox1 displayed a white dense mycelium lacking acervuli, accompanied by a 96% reduction in conidial number. A significant reduction in the rate of mycelial growth was observed in Δnox1 and ΔnoxR compared with the wild type. Disruption of nox2 reduced disease severity in daughter plants as opposed to that for the Δnox1 and ΔnoxR strains. Disruption of nox1 resulted in a significant decrease in ROS production in conidia, before and during germination. Disruption of nox2 was not accompanied by reduced ROS production in non-germinated conidia, yet resulted in elevated ROS levels in germinating conidia. ΔnoxR disruption displayed reduced ROS production in non-germinated conidia and elevated ROS levels in germinating conidia. We concluded that: (i) nox1 plays a key role in acervulus and conidial differentiation; (ii) nox2 is probably involved in the early stage of infection on strawberry plants; and (iii) for the first time in an ascomycete, it appears that noxR is not essential for nox1 activity. Identification of the ROS species generated by germinating conidia of Δnox2 or ΔnoxR may elucidate the nature of possible cross-talk between different fungal ROS generating pathways.

Exploiting the potential of pattern recognition receptors to improve crop resistance

O. Bahar^1,*, B. Schwessinger¹, N. Thomas¹, J.N. Tripathi², J. Lorenzen³, V. Nekrasov⁴, C. Zipfel⁴, L. Tripathi² and P. Ronald¹

¹ Dept. of Plant Pathology and the Genome Center, University of California Davis, Davis, CA 95616, USA [^* e-mail: ofirb@volcani.agri.gov.il]; ² International Institute of Tropical Agriculture (IITA), Nairobi, Kenya; ³ International Institute of Tropical Agriculture (IITA), Arusha, Tanzania; and ⁴ The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK

Innate immunity is the first line of induced defense against pathogen attack and is rapidly activated following infection. Conserved molecules exposed by pathogens are recognized by plant plasma membrane-localized pattern recognition receptors (PRRs) that initiate intracellular signaling leading to broad-spectrum disease resistance. Unlike typical resistance genes (R genes), which recognize unique molecules that are, in most cases, dispensable for pathogen fitness, PRRs recognize conserved microbial molecules essential for pathogen fitness. Therefore, these receptors serve as good candidates to provide improved and long-lasting disease resistance. Here we present two examples of PRR transfer between species to improve disease resistance: (i) the transfer of the bacterial elongation factor (EF-Tu) receptor (EFR) from Arabidopsis to rice; and (ii) the transfer of rice XA21 to banana. Transgenic rice lines expressing the EFR receptor became fully responsive to bacterial EF-Tu, demonstrating the feasibility of transferring functional PRRs from dicotyledonous to monocotyledonous species. EFR-expressing rice lines became more tolerant to Xanthomonas oryzapv . oryzae infection. Banana plants expressing the rice XA21 receptor became fully resistant to the banana Xanthomonas wilt pathogen X. campestris pv. musacearum, thereby providing a valuable tool for controlling the pandemic of Xanthomonas wilt disease of banana – a staple food for 100 million people in east Africa. In conclusion, these two examples demonstrate the practicability of engineering PRRs into important crop plants to improve disease resistance.

Characterization of Aegilops speltoides introgression lines conferring resistance to wheat leaf rust

J. Deek* and A. Distelfeld

Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 6997801, Israel [^* e-mail: jaslineba@gmail.com]

Bread wheat (Triticum aestivum L.) is the most widely grown crop in the world and provides 20% of the daily protein and calories for humanity. To meet the growing food demand, annual wheat yield increases must rise by at least 2%. Therefore, great efforts must be made to increase wheat yield, and tolerance to abiotic stresses, pathogens and pests. Leaf rust (Puccinia triticina) is the most widely distributed disease of wheat, causing an average annual yield loss of 3% worldwide. Rust resistance genes that have been incorporated into modern cultivars, namely, host resistance, is the most effective and economical method of disease control. Here we report the phenotypic and molecular characterization of wheat (Aegilops speltoides) introgression lines (ILs) carrying a leaf rust resistance. The use of molecular markers revealed three main introgressions from Ae. speltoides to the elite cultivar 'Barnir' background. To further reduce the introgressions, we have backcrossed the resistant ILs to 'Barnir'. The phenotypic and molecular data from the BC₁F₂ segregating ILs suggest that the resistance is genetically transmitted by a single dominant gene located in a 60 cM region on chromosome 1B. The molecular markers which are tightly linked to the smaller introgression involving the resistance can be used to transfer the resistance gene to new wheat varieties by marker-assisted breeding. The newly developed ILs are currently being tested in the field to evaluate their agronomic performance and rule out possible linkage drags.

The specificity of Blumeria graminis tritici virulence, at the primary center of origin of wheat, is determined by the host of the mildew

R. Ben-David¹, E. Kosman², R. Parks³, C. Cowger², T. Wicker⁴, A. Dinoor⁵ and B. Keller⁴

¹ Dept. of Vegetables and Field Crops, ARO, The Volcani Center, Bet Dagan 50250, Israel [*e-mail: roib@volcani.agri.gov.il]; ² Institute for Cereal Crops Improvement (ICCI), The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel; ³ U.S. Department of Agriculture–Agricultural Research Service, Dept. of Plant Pathology, North Carolina State University Raleigh, NC, 27695, USA; ⁴ Dept. of Plant Biology, University of Zurich, 8008 Zurich, Switzerland; and ⁵ Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

A collection of Blumeria graminis f.sp. tritici (Bgt) isolates was established by N. Eshed in 1985, and is maintained and studied by A. Dinoor. In 1994 Eshed and colleagues concluded that, based on host reactions, there are two types of Bgt: one collected from domesticated wheat (Triticum aestivum and T. durum), which is virulent on T. aestivum, T. durum and T. dicoccoides; and a second, collected from wild emmer wheat, which is virulent only on tetraploid wheats. The current research included 61 Bgt isolates from those three host species collected from four eco-geographic regions within Israel. The phenotypic characterization of the Bgt collection was performed in virulence tests on 16 wheat lines. The virulence scores were analyzed using the software Virulence Analysis Tool (VAT). The molecular assays included seven simple sequence repeat (SSR) markers and genomic genic and inter-genic sequences. In general, all assays divide the collection into two distinct groups based on the hosts of the isolates: domesticated hosts (T. durum and T. aestivum) and wild hosts (T. dicoccoides) from natural habitats. This pattern was consistent in the three analyses (i.e., virulence assay, SSR markers and sequence diversity). In addition, we identified some outlier genotypes (e.g. based on the SSR assay, some Bgt isolates sampled from wild T. dicoccoides plants were grouped together with those collected from domesticated wheat). Such outliers may point to a possible gene flow between cultivated wheat fields and wild wheat in natural populations. The eco-geographical factor, however, was not significant.

Posters presented at the 35th Congress of the Israeli Phytopathological Society

Effects of fertilization regimes on bacterial fruit blotch disease of melon caused by Acidovorax citrulli

N. Zimerman^1,*, M. Shenker² and S. Burdman¹

¹ Dept. of Plant Pathology and Microbiology and ² Dept. of Soil and Water Sciences, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel [*e-mail: naama.zimerman@mail.huji.ac.il]

Bacterial fruit blotch of cucurbits, caused by Acidovorax citrulli, is a destructive disease that threatens the melon and watermelon industries worldwide. Finding a fertilizer regime that reduces the establishment of A. citrulli during plant growth may contribute to bacterial fruit blotch management and to a reduction of yield losses. Several experiments were performed in which we tested various levels of nitrogen (N) and potassium (K) in different combinations. Three- to four-week-old melon plants were inoculated with A. citrulli. We assessed disease symptoms and the A. citrulli population on leaves for a period of 4 weeks after inoculation. For N fertilization we tested three solutions having different ratios of ammonium (NH₄ ⁺) and nitrate (NO₃ ^-). The intermediate solution, which contained a 80:20 nitrate:ammonium ratio resulted in the highest disease severity, while fertilization with 100% NO₃ ^- was associated with reduced levels of disease severity. Increased K concentrations in the fertilization solution were also associated with decreased disease severity, as well as with a significant reduction of the A. citrulli population in infected leaves. RNA extraction from leaves was performed at several times after inoculation to assess expression of plant defense genes and A. citrulli virulence genes by qRT-PCR. These experiments are proceeding and results will be presented in the future.

An efficient method for the production of pathogen-free potato (Solanum tuberosum) plants

Y. Tam*, S. Singer and V. Gaba

Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250, Israel [*e-mail:yehudit@volcani.agri.gov.il]

Potato (Solanum tuberosum) is the fourth most important food crop worldwide, the largest by volume in Israel, and is very susceptible to disease. Production of pathogen-free potato stocks begins with the tissue culture of disease-indexed plants. However, potato plants in vitro exhibit severe symptoms of excessive ethylene accumulation. Plants of normal appearance can be produced in vitro (cvs. ‘Nicola’, ‘Ratte’, ‘Desirée’, ‘Claustar’, ‘Maris Peer’) by adding silver thiosulfate (2 mg l ^-1) to Murashige and Skoog (1962) basal medium and sealing the test tubes with ‘Steristopper’ breathable cellulose plugs. Several cultivars have thus been maintained in vitro (20 μmol.m^-2s^-1 PAR fluorescent cool-white light, 16-h photoperiod, 23–25°C) for 10 years, subcultured every 4–6 weeks, with a multiplication factor of 4–6. Hardening of plants is combined with multiplication. Shoot sections bearing axillary buds and expanded green leaves are excised from in vitro-grown potato plantlets, and planted directly into rockwool plugs, in 0.5 or 1 liter polypropylene, translucent, disposable food-grade containers. The plugs are soaked in distilled water and 1 g of Osmocote slow-release fertilizer is added. After planting, containers are covered with a re-usable lid with a large filter-paper-covered hole and placed in a growth room with 100–120 μmol.m^-2s^-1 PAR fluorescent cool white light, 16-h photoperiod, 23–27°C. After 5 days the cuttings have rooted, the lids are removed, the plants continue to grow autotrophically, and are ready for transfer after 3.5 weeks. Nearly 100% of cuttings produce healthy plants, which can be transferred directly to the greenhouse for minituber production.

Characterization of the Grapevine virus A AlkB motif and its involvement in plant viral infection

C. Dayan-Glick*, L. Maslenin, Y. Iddan and M. Mawassi

Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250, Israel [^* e-mail: *kathy@volcani.agri.gov.il]

Grapevine is economically important and is one of the most widely grown fruit crops worldwide. Viral diseases constitute a major hindrance to the development of highly profitable production of viticulture. Rugose wood (RW) complex diseases cause serious damage to grapevine. Grapevine virus A (GVA), genus Vitivirus, family Betaflexiviridae, is closely associated with RW complex diseases. The viral positive single-stranded RNA genome consists of five open reading frames (ORFs). ORF1 encodes replication-associated proteins and a protein with an AlkB motif. AlkB proteins have homologs in multicellular organisms, and are involved in the mechanism of nucleic acid de-methylation. However, the specific role of AlkB in viruses is still unknown. In this research we have utilized GVA- and AlkB-mutated derived GVA clones to examine involvement of the AlkB domain in plant infection, as well as in suppression of RNA silencing. Nicotiana benthamiana plants agro-inoculated with some GVA variants with a mutated AlkB domain showed late symptom development. In grapevine, we found that GVA variants with a mutated AlkB domain can inoculate plantlets, but cannot survive for a long time in mature plants. ORF5 encodes a small protein, P10, which exhibits weak RSS activity. We found that expression of AlkB enhances P10 RSS activity. However, AlkB mutants which were late in symptom development also suppressed P10 and AlkB RSS co-activity. Using the Yeast Two Hybrid system we found that P10 is capable of interacting with AlkB protein and exhibits self-interaction.

Highly effective technique to induce indirect death of plant pests and plant diseases

S. Shay^1,* and D. Talmor²

¹ Sorbonne University and Green Life Development, Rishon leZiyyon 75319 [*e-mail: sam@greenlifedevelopment.co.il]; and ² Purdue University and Green Life Development, Rishon leZiyyon 75319, Israel

A major challenge for global agriculture is the damage caused to plants by pests and diseases. Many of the methods used in the attempt to control agricultural pests and diseases are chemicals or other materials of themselves dangerous for people, animals and the environment. As part of an undertaking related to fat molecules for the purpose of improving the effectiveness of cleaning products, it was discovered that the manipulation of oil molecule chains can affect fat molecules in the joints of plant pests (such as in the legs, wings, or jaws) and in the bacterial capsule, with the effect of disabling/immobilizing the pest or exposing the bacterium and leading to the death of the pest/bacterium. Further enhancement of the oil molecule manipulation produces the effect of neutralization by pepsin in human and animal digestive systems and by chlorophyll in plants, making this enhancement completely harmless to humans, animals and plants. Further research into the primary and secondary impacts of the oil molecule manipulation is proposed.

Posters Presented at the 34th Congress of the Israeli Phytopathological Society

Involvement of regulatory systems in gall formation by Pantoea agglomerans pv. betae

O. Dror^1*, L. Chalupowicz¹, G. Szhafir², M. Panijel², I. Barash² and S. Manulis-Sasson¹

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: oritd@volcani.agri.gov.il]; and ² Dept. of Plant Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel

Pantoea agglomerans pv. betae (Pab) is a gall-forming pathogen on beet and gypsophila. Pathogenicity is dependent on a functional hrp/hrc gene cluster, genes encoding for type III effectors, and a cluster of genes encoding for biosynthesis of phytohormones. A quorum sensing (QS) regulatory system and a Gac/Rsm global regulatory pathway have been characterized recently in Pab. The goal of the present study was to characterize these two systems in Pab and evaluate their effect on pathogenicity in beet and the interaction with the Hrp regulon. The genes responsible for QS in Pab were identified and include pabI, encoding the synthase for production of the N-butanoyl-L-homoserine lactone (C₄-HSL), and pabR encoding the transcriptional regulator. Pathogenicity tests conducted on leaves and cubes of table beet showed that mutants in pabI and pabR caused a significant decrease in gall size. The Gac/Rsm pathway, namely, the GacS/GacA two-component system, the transcriptional regulator RsmA and its sRNA antagonist rsmB, have been shown to be involved in the control of the Hrp regulon in Pab. Insertional mutants in gacS, gacA or over-expression of rsmA caused a substantial decrease in gall size, while over-expression of rsmB nearly abolished gall formation. By employing the ice nucleation (inaZ) reporter gene, it was shown that pabR acts directly as the transcriptional activator of hrpL, a central gene of the Hrp regulatory cascade. In addition, a mutant in pabR substantially reduced the transcriptional level of hrpL as determined by qRT-PCR in planta. Results suggest that the interplay between the QS system, the Hrp regulatory cascade and the Gac/Rsm pathway control the pathogenicity of Pab.

Growth parameters in plants propagated from grapevine recovered from yellows disease

E. Gol¹, A. Vasserman¹, M. Harel¹, M. Dafni-Yelin^1,2, T. Zehavi³ and V. Naor^1,2,*

¹ School of Oenology Studies, Ohalo College, Qazrin 12900; ² Golan Research Institute – Haifa University, Qazrin 12900 [*e-mail: vered_n@macam.ac.il]; and ³ Extension Service, Ministry of Agriculture and Rural Development, Qiryat Shemona 10200, Israel

Yellows disease in grapevine is caused by phytoplasma resulting in reduced biomass and yield. In certain cultivars symptom remission and a gradual increase in plant biomass and yield are observed in the year following infection. This phenomenon, known as recovery, is reported in grapevines and apples and persists for 10–12 years. However, it is not clear whether it is possible to exploit the recovery phenomenon to produce ‘resistant’ plant material. Furthermore, the quality of recovered vines as a source for propagation is not known. The aim of this work was to compare growth parameters of plants propagated from recovered and healthy vines. Scions of cv. ‘Cabernet Sauvignon’ were grafted on ‘Richter 110’ rootstock in the winter of 2010. The plants were grown in an insect-proof net-house in 5 l pots and drip irrigated. Bud sprouting, cane diameter at the basipetal end, total cane length, prune weight and number of clusters and berry size were measured. The results indicated similar growth performance of plants propagated from both vine types. Plants from healthy vines sprouted a little earlier than those from recovered vines. Shoot diameter and total cane length of plants from recovered vines were similar to plants from healthy vines. Prune weight of plants from recovered vines was 15% higher. No differences were found in the number of clusters, but plants from recovered vines had larger berries. Thus, if recovered vines are proved to be more resistant to re-infection it may be possible to use these plants as propagation material. This issue needs further study.

Fungicide resistance in Botrytis cinerea collected from sweet basil crops

N. Korolev^1,*, M. Mamiev¹, S. Biton², D. Silverman² and Y. Elad¹

¹ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250 [*e-mail: vpptlg@volcani.agri.gov.il]; and ² Extension Service, Ministry of Agriculture and Rural Development, Bet Dagan 50250, Israel

A total of 648 Botrytis cinerea isolates were collected from diseased sweet basil plants and the air in ten sweet basil greenhouses. Mycelial growth tests were used to evaluate the sensitivity of these isolates to polyoxin AL (the main botryticide on sweet basil in Israel) and other fungicides. Among populations that had not been exposed to polyoxin treatments, 20– 35% of the collected isolates were low-level resistant to polyoxin. Polyoxin treatments shifted the equilibrium in favor of low-level resistant isolates, to a frequency of 72% after a few treatments over two seasons. Prolonged use of polyoxin in Israeli basil crops (in some sites for more than 10 years) does not appear to have led to the development of high-level resistance, but low-level resistant isolates were found in commercial greenhouses with a frequency of up to 73%. High-level resistance to benzimidazoles was common (60–80% of isolates) in greenhouses with a history of benzimidazole treatments, whereas 15–25% of the isolates from greenhouses in which fungicides were not used were resistant. Low-level resistance to dicarboximides was fairly widespread (30–80%) and a few cases of moderate resistance to dicarboximides were also noted (up to 9%). Neither high- nor low-level resistance to anilinopyrimidines was common in commercial greenhouses (up to 7%); however, 34% of the isolates were strongly resistant in the experimental greenhouse, following a few treatments with anilinopyrimidine fungicides during the previous season. About 3% of the isolates exhibited low-level resistance to fenhexamid and no isolates were found to be strongly resistant to fenhexamid. Treatments with Signum (premixed formulation of boscalid and pyraclostrobin) in two sweet basil commercial greenhouses during one season, caused the rapid development of resistance with 53% of dual resistant isolates in one greenhouse and 100% of dual resistant isolates in the other one.

Dematophora necatrix in deciduous orchards: Evaluation of pathogen distribution and a protection strategy to prevent additional spread

S. Nasralla¹, A.M. Safadi², P. Safadi³, S. Freeman⁴, M. Taiseer¹, S. Kfir⁵, O. Levi⁵, M. Miron⁵ and M. Dafny-Yelin^5,6,*

¹ Elmagareq Agricultural Cooperative, Majdel Shams 12438; ² El-Sharaq Agricultural Cooperative, Majdel Shams 12438; ³ Saar Agricultural Cooperative, Mas`ade 12438; ⁴ Dept. of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan 50250; ⁵ MIGAL-Northern Research & Development, Qiryat Shemona; and ⁶ Golan Research Institute – Haifa University, Qazrin 12900, Israel [*e-mail: merydy@gmail.com]

White root rot disease caused by the fungus Dematophora necatrix is destructive to many fruit trees, particularly apple and other deciduous crops. The symptoms include rotting of the roots and yellowing of leaves, followed by wilting and tree death. In the north of Israel farmers have reported widespread wilting of deciduous trees, but the origin and true impact of this problem is poorly understood. In many cases, soilborne pathogens were thought to be the problem. Today, there is no treatment for Dematophora infection in Israel, and the disease spreads continuously every year to new trees. The research objectives were: evaluation of damage due to Dematophora, and application of fungicide against disease spread. During the study, 120 root samples were collected from suspect diseased orchards, washed and kept in a moist chamber. After a few days microscopic examination confirmed the appearance of typical mycelium of the pathogen. Dematophora root infection was found in 27 plots: 15 in rural areas, nine in Metulla, and single plots in Jish, Netu`a, and Margaliot. In addition, Sclerotium rolfsii was found in eight locations. Fungicide treatments showed that carbendazim, fluazinam and thiophanate-methyl inhibited fungus growth on PDA medium and prevented plant wilting in artificial pot inoculation trials. The ability of these fungicides to inhibit disease spread to neighboring trees in infected orchards is still under study. The results show that Dematophora is spreading into new areas in northern Israel, and may be controlled with the use of fungicides.