Identification and genetic mapping of PmAF7DS a powdery mildew resistance gene in bread wheat (Triticum aestivum L.).

KEY MESSAGE: Gene PmAF7DS confers resistance to wheat powdery mildew (isolate Bgt#211 ); it was mapped to a 14.6-cM interval ( Xgwm350 a- Xbarc184 ) on chromosome 7DS. The flanking markers could be applied in MAS breeding. Wheat powdery mildew (Pm) is caused by the biotrophic pathogen Blumeria graminis tritici (DC.) (Bgt). An ongoing threat of breakdown of race-specific resistance to Pm requires a continuous effort to discover new alleles in the wheat gene pool. Developing new cultivars with improved disease resistance is an economically and environmentally safe approach to reduce yield losses. To identify and characterize genes for resistance against Pm in bread wheat we used the (Arina × Forno) RILs population. Initially, the two parental lines were screened with a collection of 61 isolates of Bgt from Israel. Three Pm isolates Bgt#210 , Bgt#211 and Bgt#213 showed differential reactions in the parents: Arina was resistant (IT = 0), whereas Forno was moderately susceptible (IT = -3). Isolate Bgt#211 was then used to inoculate the RIL population. The segregation pattern of plant reactions among the RILs indicates that a single dominant gene controls the conferred resistance. A genetic map of the region containing this gene was assembled with DNA markers and assigned to the 7D physical bin map. The gene, temporarily designated PmAF7DS, was located in the distal region of chromosome arm 7DS. The RILs were also inoculated with Bgt#210 and Bgt#213. The plant reactions to these isolates showed high identity with the reaction to Bgt#211, indicating the involvement of the same gene or closely linked, but distinct single genes. The genomic location of PmAF7DS, in light of other Pm genes on 7DS is discussed.

Additional Vegetative Compatibility Groups in Colletotrichum coccodes Subpopulations from Europe and Israel.

Potato black dot, caused by Colletotrichum coccodes, damages tuber quality and may reduce yield. In previous work, four multimember vegetative compatibility groups (VCGs) have been reported. The objectives of the current study were to characterize a population of C. coccodes comprised of isolates from Israel and Northern Europe (EU/I) using VCG, and to assess the correlation between VCGs and aggressiveness of isolates on potato. A composite of 176 isolates was collected from symptomatic tissues of potato tubers or stems. A total of 6 (3.4%) isolates were characterized in VCG1; 29 (16.5%), 32 (18.2%), and 7 (4.0%) in VCG 2, 3, and 4, respectively; and 7 (4.0%), 9 (5.1%), 48 (27.3%), and 15 (8.5%) in the newly defined VCG 5, 6, 7, and 8, respectively. Twenty-three isolates (13%) were not assigned to any of the VCGs. Two of the VCGs had a specific geographical distribution: the 9 isolates assigned to VCG6 originated from The Netherlands, and 34 of 38 isolates assigned to VCG7 were from Scotland. Aggressiveness of isolates of a given VCG was examined on potato. VCGs 5 and 6 were comprised of the most aggressive isolates, and VCG1 of the least aggressive. These results could facilitate a more accurate evaluation of damage potential that may be caused by this pathogen.

Production of Didymella rabiei Pseudothecia and Dispersal of Ascospores in a Mediterranean Climate.

ABSTRACT Temperature and wetness conditions required for development and maturation of Didymella rabiei pseudothecia were determined in a series of experiments conducted in controlled-environmental conditions. Initial stages of pseudothecium formation occurred at temperatures ranging from 5 to 15 degrees C. Incubation at low temperatures was essential for subsequent pseudothecium maturation. This requirement was satisfied for chickpea stem segments incubated at 5 or 10 degrees C for three consecutive weeks or during periods of 3 or 5 days, separated by periods at higher temperatures. Following the low-temperature requirement, subsequent pseudothecium development was independent of temperature in the range tested (5 to 20 degrees C). Wetness was essential for pseudothecium production: pseudothecia formed and matured on stem segments maintained continuously wet but also on those exposed to periods of three or five wet days, separated by dry periods. The dispersal of D. rabiei ascospores was studied using chickpea plants as living traps in the field. Trap plants were infected mainly when exposed during rain but also in rainless periods. Results of this study enabled us to describe the developmental events leading to the production of the teleomorph stage and the dispersal of ascospores by D. rabiei in the Mediterranean climate of Israel.

Significance of Preventing Primary Infections by Didymella rabiei and Development of a Model to Estimate the Maturity of Pseudothecia.

The significance of preventing primary infections resulting from the teleomorph stage of Didymella rabiei was tested in field experiments in 1998 and 2000. Control efficacy was greater and yield and its components were higher in plots where the fungicide difenoconazole had been sprayed in time to protect the plants from infections resulting from airborne ascospores than in plots where sprays were not applied on time. Forty empirical models reflecting the influence of temperature and interrupted wetness on initial maturation of D. rabiei pseudothecia were developed and verified by using data recorded in chickpea fields in 1998. Seven of the models then were validated with data recorded in 1999 and 2000. The following model provided the best predictions: starting at the beginning of the rainy season (October to December), the predictor of the model was assigned one severity value unit when there was a rain event (1 day or more) with ≥10 mm of rain and an average daily temperature (during the rainy days) of ≤15°C. According to the model, pseudothecia mature after accumulation of six severity values and ascospores will be discharged during the following rain.

External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides.

Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichum gloeosporioides: As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased. Nitrogen assimilation also was required for PL secretion at pH 6.0. Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0. Sequence analysis of the 5' upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC. Consistent with this result, the transcript levels of pac1 (the C. gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH. Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C. gloeosporioides.

Joint action of disease control measures: a case study of alternaria leaf blight of carrot.

ABSTRACT The efficacy of chemical (i.e., foliar fungicide sprays), genetic (i.e., moderately resistant cultivars), and cultural (i.e., drip-irrigation system) control measures was quantified individually and in combination in the management of Alternaria dauci, the causal agent of Alternaria leaf blight of carrot. Whereas host resistance and drip irrigation affected both the time of disease onset and the rate of disease progression, chemical control affected only the latter. In all cases, a single control measure did not provide an acceptable level of disease suppression. Control efficacy values (based on the relative area under the disease progress curve) for chemical, genetic, and cultural control were 58 +/- 11, 39 +/- 20, and 60 +/- 22%, respectively (values are means +/- standard error). By contrast, implementing two control measures concurrently always improved disease suppression significantly compared with the individual measures. Control efficacy values were 91 +/- 8% for the integration of chemical and genetic measures and 82 +/- 23% for the integration of chemical and cultural measures. Moreover, yields in plots protected by two control measures simultaneously were higher by 10.1 to 28.6 t/ha than those in the respective plots protected by single measures. The joint effect of chemical control and host resistance was additive, whereas that of chemical control and drip irrigation was synergistic in most cases. A literature review was performed to determine if these findings represent a general relationship between chemical and genetic, and chemical and cultural measures. Based on 19 reviewed cases, it was concluded that additive effects are the rule and synergistic or antagonistic effects are the exception. Synergistic effects of two control measures were observed when one control measure improved the efficacy of the other directly or when one control measure induced host resistance or predisposed the pathogen to increased susceptibility. These results may enable a more effective selection of candidate control measures for integration in the future.

Improving biological control by combining biocontrol agents each with several mechanisms of disease suppression.

ABSTRACT Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves and plants. Scanning electron microscopy revealed significant inhibition of Botrytis cinerea conidial germination in the presence of Pichia guilermondii, whereas Bacillus mycoides caused breakage and destruction of conidia. When both biocontrol agents were applied in a mixture, conidial destruction was more severe. The modes of action of each of the biocontrol agents were elucidated and the relative quantitative contribution of each mechanism to suppression of Botrytis cinerea was estimated using multiple regression with dummy variables. The improvement in control efficacy achieved by introducing one or more mechanisms at a time was calculated. Pichia guilermondii competed with Botrytis cinerea for glucose, sucrose, adenine, histidine, and folic acid. Viability of the yeast cells played a crucial role in suppression of Botrytis cinerea and they secreted an inhibitory compound that had an acropetal effect and was not volatile. Bacillus mycoides did not compete for any of the sugars, amino acids, or vitamins examined at a level that would affect Botrytis cinerea development. Viable cells and the compounds secreted by them contributed similarly to Botrytis cinerea suppression. The bacteria secreted volatile and non-volatile inhibitory compounds and activated the defense systems of the host. The nonvolatile compounds had both acropetal and basipetal effects. Mixture of Pichia guilermondii and Bacillus mycoides resulted in additive activity compared with their separate application. The combined activity was due to the summation of biocontrol mechanisms of both agents. This work provides a theoretical explanation for our previous findings of reduced disease control variability with a mixture of Pichia guilermondii and Bacillus mycoides.

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Combining biocontrol agents to reduce the variability of biological control.

ABSTRACT Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves. The aims of the research were to determine whether the use of their combination would broaden the environmental conditions under which biological control is effective, and to test the hypothesis that it would reduce the variability of control efficacy under diverse conditions. Applied separately, the biocontrol agents significantly inhibited spore germination, lesion formation, and lesion development at most temperatures, relative humidities, and spray-timing combinations (temperatures: 10, 15, 20, 23, 25, and 30 degrees C; relative humidities: 78, 85, 96, and 100%; and spray-timings: 0, 4, and 7 days before inoculation). However, control efficacy was highly variable, and under certain combinations it was not adequate. Control efficacy achieved by the biocontrol agents applied separately ranged between 38 and 98% (mean 74%) and the coefficient of variation ranged from 9.7 to 75%. The mixture of Bacillus mycoides and Pichia guilermondii suppressed Botrytis cinerea effectively (80 to 99.8% control) under all conditions, and the coefficients of variation were as low as 0.4 to 9% in all cases. Thus, application of both biocontrol agents resulted in better suppression of Botrytis cinerea, and also reduced the variability of disease control. Application of more than one biocontrol agent is suggested as a reliable means of reducing the variability and increasing the reliability of biological control.

Development of Colletotrichum gloeosporioides Restriction Enzyme-Mediated Integration Mutants as Biocontrol Agents Against Anthracnose Disease in Avocado Fruits.

ABSTRACT Reduced-pathogenicity mutants of the avocado fruit pathogen Colletotrichum gloeosporioides isolate Cg-14 (teleomorph: Glomerella cingulata) were generated by insertional mutagenesis by restriction enzyme-mediated integration (REMI) transformation. Following seven transformations, 3,500 hygromycin-resistant isolates were subjected to a virulence assay by inoculation on mesocarp and pericarp of cv. Fuerte avocado fruits. Fourteen isolates showed a reduced degree of virulence relative compared with wild-type Cg-14. Two isolates, Cg-M-142 and Cg-M-1150, were further characterized. Cg-M-142 produced appressoria on avocado pericarp similar to Cg-14, but caused reduced symptom development on the fruit's pericarp and mesocarp. Isolate Cg-M-1150 did not produce appressoria; it caused much reduced maceration on the mesocarp and no symptoms on the pericarp. Southern blot analysis of Cg-M-142 and Cg-M-1150 showed REMI at different XbaI sites of the fungal genome. Pre-inoculation of avocado fruit with Cg-M-142 delayed symptom development by the wild-type isolate. Induced resistance was accompanied by an increase in the levels of preformed antifungal diene, from 760 to 1,200 mug/g fresh weight 9 days after inoculation, whereas pre-inoculation with Cg-M-1150 did not affect the level of antifungal diene, nor did it delay the appearance of decay symptoms. The results presented here show that reduced-pathogenicity isolates can be used for the biological control of anthracnose caused by C. gloeosporioides attack.

Optimization of Chemical Suppression of Alternaria dauci, the Causal Agent of Alternaria Leaf Blight in Carrots.

Alternaria leaf blight, caused by Alternaria dauci, is a major constraint to carrot production in Israel. Israeli carrot growers apply prophylactic sprays at 3- to 10-day intervals throughout the season until harvest, up to 30 sprays in a growing season. In this study, we attempted to optimize the chemical suppression of the disease, in order to reduce fungicide use. The efficacy of nine fungicides was determined in two field experiments. All fungicides reduced disease severity, but there were significant differences in efficacy among them. The most effective were difenoconazole and chlorothalonil; less effective were copper hydroxide, tebuconazole, trifloxystrobin, and mancozeb; the least effective in our experiments were flutrifol, propineb, and iprodione. The effect of the time of spray initiation on fungicide efficacy was determined in three field experiments. Qualitative (analysis of variance) and quantitative (regression) analyses of the data revealed that initiating sprays after disease onset reduced control efficacy. Thus, an action threshold model could not be developed for A. dauci in carrots. The time before harvest at which sprays could be terminated was tested in two field experiments and it was found that terminating sprays 14 days before harvest did not significantly affect the overall control efficacy. The main conclusions derived from these experiments were tested and corroborated in two additional field experiments.

Expression of pectate lyase from Colletotrichum gloesosporioides in C. magna promotes pathogenicity.

To test the contribution of pectate lyase (PL) to promoting fungal pathogenicity, a pectate lyase gene (pel) from the avocado pathogen Colletotrichum gloeosporioides, isolate Cg-14, was expressed in C. magna isolate L-2.5, a pathogen of cucurbits that causes minor symptoms in watermelon seedlings and avocado fruits. Isolate L-2.5 was transformed with pPCPH-1 containing hph-B as a selectable marker and the 4.1-kb genomic pel clone. Southern hybridization, with the 4.1-kb genomic pel clone or 2.13-kb hph-B cassette as probes, detected integration of pel in transformed C. magna isolates Cm-PL-3 and Cm-PL-10. Western blot (immunoblot) analysis with antibodies against Cg-14 PL detected a single PL secreted by L-2.5 at a molecular mass of 41.5 kDa, whereas the PL of C. gloeosporioides had a molecular mass of 39 kDa. When PL activity was measured 4 days after inoculation in pectolytic enzyme-inducing media (PEIM), transformed isolates Cm-PL-3 and Cm-PL-10 showed additive PL activity relative to both Cg-14 and L-2.5. Transformed isolates also showed additive maceration capabilities on avocado pericarp relative to the wild-type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, more severe maceration and damping off developed in watermelon seedlings inoculated with the transformed isolates compared with the two wild-type isolates, which showed no symptom development on these seedlings during the same period. Results clearly show the contribution of a single pel to the pathogenic abilities of C. magna and suggest that PL is a pathogenicity factor required for the penetration and colonization of Colletotrichum species.

pH regulation of pectate lyase secretion modulates the attack of Colletotrichum gloeosporioides on avocado fruits.

Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3. 8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), where pg transcripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to C. gloeosporioides attack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affect C. gloeosporioides pathogenicity. The mechanism found in avocado may have equivalents in other post-harvest pathosystems and suggests new approaches for breeding against and controlling post-harvest diseases.

Resistance of Gibberellin-Treated Persimmon Fruit to Alternaria alternata Arises from the Reduced Ability of the Fungus to Produce Endo-1,4-beta-Glucanase.

ABSTRACT Black-spot symptoms, caused by Alternaria alternata, developed in persimmon fruits during prolonged storage at -1 degrees C. A preharvest treatment with gibberellic acid (GA(3)) extended the storage life of the fruit by delaying both black-spot development and fruit softening. Conversely, treatment of persimmon fruits with paclobutrazol (PBZ), an inhibitor of gibberellin (GA) synthesis, enhanced black-spot development and fruit softening during storage. Production of endo-1,4-beta-glucanase (EC 3.2.1.4, EG) by A. alternata in culture and in the presence of cell walls from PBZ-treated fruits as the carbon source, was enhanced by 150% over production in the presence of cell walls from control fruits, whereas endoglucanase (EG) production in the presence of cell walls from GA(3)-treated fruits was reduced by 49% relative to controls. To determine the importance of EG in symptom development, A. alternata EG was purified from a culture-inducing medium. It had a molecular mass of 41 kDa, its optimal pH and temperature for activity were 5.5 and 47 degrees C, respectively, and the pI was 4.3. Its K(m) and V(max) were 0.43 mg ml(-1) and 18 mumol reducing groups minute per milligrams of protein, respectively. The internal sequence of a 21-mer amino acid peptide from the purified EG showed 62% similarity and 38% identity to the EG-1 of Trichoderma reesei and of T. longibrachiatum. Purified EG induced black-spot symptoms on the fruit, similar to those caused by A. alternata, whereas boiled enzyme caused only pricking signs. Our results suggest that the black-spot symptoms caused by A. alternata, in persimmon, are related to the ability of the fungus to produce EG in developing lesions.

Epidemiology of Botrytis cinerea in Sweet Basil and Implications for Disease Management.

Sweet basil (Ocimum basilicum) is a herbaceous annual plant that is highly susceptible to gray mold, caused by Botrytis cinerea. Infections are initiated on the surface of stem wounds that are caused during harvest; the pathogen then progresses on the stem, killing leaves and secondary buds. When the infection reaches the main stem, the entire plant dies. A study of the epidemiology of the disease and of host-pathogen interactions led to the development of rules for effective disease management. The research was conducted in three steps. (i) Natural epidemics in commercial crops were monitored during the 1993 to 1994 and 1994 to 1995 growing seasons. Disease outbreaks were found to coincide with harvests during rainy days, whereas disease intensity did not change much when harvests were completed during rainless days. (ii) Studies under controlled environmental conditions revealed that cut ends of stems were highly susceptible soon after harvest, susceptibility diminished gradually, and stem cuts inoculated 48 h after harvest were rarely diseased. Observations under a scanning electron microscope showed that an opaque layer had appeared over the cut surface and, as the entire wound surface became covered, penetration of the fungus into the tissue was prevented. Based on these findings, it was hypothesized that avoidance of harvesting during rain events and application of one fungicidal spray, soon after harvest, would result in adequate disease suppression. (iii) These hypotheses were tested and corroborated in greenhouses in two experiments, in 1994 to 1995 and 1995 to 1996. Management of basil crops according to these concepts would also lower the risks for contamination of the marketable product with pesticide residues.

Purification of pectate lyase produced by Colletotrichum gloeosporioides and its inhibition by epicatechin: a possible factor involved in the resistance of unripe avocado fruits to anthracnose.

Pectate lyase (PL) from Colletotrichum gloeosporioides was purified to apparent homogeneity by hydrophobic interaction chromatography followed by isoelectric focusing. The purified preparation showed one band corresponding to 40 kD on sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the enzyme was 7.9, and the optimum pH for activity was 8.9. The purified PL efficiently macerated unripe avocado fruit wedges. In vitro translation of mRNA from an induced fungal culture revealed a 36-kD precursor polypeptide, which was precipitated with PL antibodies. The antibodies inhibited enzymatic activity and maceration ability on avocado wedges. Epicatechin, a flavan 3-ol present in the peel of unripe avocado fruit, had a Ki of 3.4 microM for inhibition of PL activity in vitro. At 20 micrograms/ml (68 microM), epicatechin reduced the enzyme's macerating ability by 64%. Since the flavan is present in unripe fruit at much higher concentrations (about 350 micrograms/g fresh weight) than the inhibitory concentrations, epicatechin may be involved in the resistance of unripe avocado fruits by inhibiting the PL activity of C. gloeosporioides.