JWSS - Journal of Water and Soil Science

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Four hundred isolates of Ascochyta rabiei (Pass.) Lab. collected from different parts of the country, such as Zarivar Marivan Lake in Kordestan Province Shabestar and Khosroshahr in Eastern Azarbaigan Province Serow, Bokan and Shahindeg in Western Azarbaigan Province Mashhad and Ilam were used in this experiment.

These isolates showed little differences in their cultural and morphological characters and pathogenic behaviors. They were categorized into 17 groups on the basis of collection regions and, based on cultural characteristics, were then reduced to 11 groups. Isolate number 16 from Mashhad showed the highest growth rate while isolate number 1 from Kordestan had the lowest growth rate.

One isolate was chosen as a representative for each group. Pathogenicity of representative isolates from each of the 11 groups were tested. Reaction type of all isolates was studied on differential hosts and one local chickpea line Jam was examined, using Reddy and Nene (1979) method. Races No. 4 and 6 were identified as the two physiologic races.

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This study was carried out on the biochemical aspects of chickpea cultivars and the genomic behavior of A. rabiei pathotypes 4 and 6 in four parts: 1) Determining the number of resistant genes in chickpea native cultivars, 2) Comparing the variation of sodium and potassium electrolytes concentrations in noninfected and infected seedling stems of resistant and susceptible cultivars, 3) Studying the effect of potassium deficiency on five differential cultivars by Hoagland nutrient culture, and 4) Using RAPD-PCR method to detect any genomic differences between the two pathotypes used in this study.

Eighteen native chickpea cultivars were chosen for this study. The result of the experiments showed that cultivar 1-60-144 possesses the highest number of resistant genes, while the others were either relatively tolerant or susceptible. The reduction of electrolytes concentration in infected cultivar ILC-1929 in comparison to resistant cultivar ILC-5928 is an indication of compatibility between the host plant and the pathogen. Increasing the level of resistance in differential cultivars and appearance of induced resistance as the result of potassium deficiency is due to the production of putrecine diamine. Eighty percent similarity of pathotypes genomic bands by using CG marker and primer 171 showed insufficient primer numbers and the necessity for using complementary methods.

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During the summer of 1998, leaf and pod blight symptoms of bean were observed in Arak, The survey in the following year showed that the disease had an increasing trend in bean-growing regions of Markazi Province. It appeared that more severe losses occurred in fields equipped with sprinkler irrigation system. Symptoms of diseased plants consisted of formation of irregular necrotic lesions on leaves surrounded by yellow halo. Under favourable conditions, these lesions gradually expanded and heavily infected leaves became blighted. The primary symptoms on infected pods were the development of water soaked spots which later turned to dark or reddish blotches. In attempts to investigate the causal agent, two types of a Gram negative bacteria with raised, yellow and translucent colonies were isolated from leaves and pods of blighted beans. All strains produced Xanthomonadin pigment and their inoculation on young bean plants induced disease symptoms similar to those observed under field conditions. Based on morphological, physiological and biochemical properties, the predominant pathogenic type was identified as Xanthomonas axonopodis pv. phaseoli. However, the other strain with the same properties, but capable of producing melanin pigment in nutrient media, was assigned to X. axonopodis pv. phaseoli var. fuscans.

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During the period from 1997 to 1998, samples of wheat leaves were collected from different wheat farms in Fars and Kohgiluyeh & Boyrahmad provinces to identify the causal agent of wheat leaf blight. On the basis of LOPAT tests and production of fluorescent pigment on KB medium, 181 bacterial isolates were recovered from the samples. Based on biochemical, physiological and pathogenicity tests, the isolates were categorized in five groups. One group was found to be Pseudomonas fluorescens (Pf) and the remaining groups were identified as P. syringae pv. syringae (Pss). The pathogenicity test indicated that Pss strains were the causal agent of bacterial wheat leaf blight in Fars and Kohgiluyeh & Boyrahmad provinces. Whole protein electrophoretic patterns were similar in Pss isolates but only a few showed small variation in some subordinated bands. Pathogenic strains of Pss were also isolated from annual and perennial weeds such as foxtail, hairy vetch, oat grass, barley grass and Cynodon dactylon in some areas including Saadat Shahr and Marvdasht. Among the wheat cultivars tested, Tajan was susceptible to the pathogen but others were immune, resistant, moderately resistant, or moderately susceptible. The results showed that Pss is a seed-born pathogen in wheat kernel perhaps as endophyte in the seeds.

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Antagonistic fungi Trichoderma harzianum (T₁, from bean fields in Ahwaz, T₂, from rice fields in Rasht), T. viride (T₃ from bean fields in Shahriar, T₄ from the collection in Plant Pest and Disease Institute, Tehran), Gliocladium virens (from bean fields in Kamal Abad, Karaj) and some fungicides (Benomyl, Carbendazim, Carboxin-Thiram, edifenphos and Zineb) were used to control sheath blight of rice incited by Rhizoctonia solani in vitro and under greenhouse conditions. The experiment was carried out in a completely randomized design with 12 treatments and four replications on Khazar rice cultivar in a soil infected by R. solani under greenhouse conditions. The results showed that the antagonistic fungi reduce sheath blight by 19.8 (T₁), 21.5 (T₂), 27.5 (T₃), 19.6 (T₄) and 18.5 (G₁) percent. The antagonistic fungi in order of efficacy were T₂>T₃>T₁>T₄>G₁. Benomyl, Carbendazim, Carboxin-thiram, Edifenphos and Zineb reduced disease by 32.5, 21.5, 12.8, 9.5 and 0 percent, respectively. Statistical analysis of data indicated that there existed no significant differences between T₁, T₃, T₄, and G₁ and Carbendazim fungicide to control disease however, the isolate T₂ was as effective as Benomyl but Zineb had no effect on sheath blight.

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The poor information available on variation of Ascochyta blight fungus is the most important factor limiting chickpea breeding programs for resistance to blight disease. In this study, efforts were made to detect genetic variation of the pathogen in Iran. The RAPD marker was employed to evaluate 26 isolates collected from 16 provinces. Twelve random primers were used to analyze genomic DNA of the isolates. Only ten primers showed polymorphism among isolates. Primer OPK-01 defined the highest number of polymorphism and primer OPK-09 confirmed relatively low degree of polymorphism. On the basis of this molecular marker, the estimated genetic diversity index was 98% and the pair-wise genetic distance of the isolates varied from 0.16 to 0.61. The least genetic distance belonged to isolates 20 and 22 from Qazvin and Golestan while the highest distance belonged to isolates 26 and 12 from Mazandaran and Markazi. The phylogenic tree was constructed by cluster analysis and all the isolates grouped to 22 genetic clusters at the 90% similarity level. The genetic groups were named from A to V and their distribution in Iran was determined. The results revealed that genetic variation among Iranian population of the pathogen is very high, and further that RAPD is a vigorous tool for genomic analysis of Ascochyta rabiei.

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The fire blight with the bacterial causal agent, Erwinia amylovora (Burrill) winslow etal. is one of the most important diseases of the pome fruits that causes the economical losses to quince, pear and apple productions, respectively, in some parts of country. To determine the infection severity of the 43 raturally infected pear cultivars in collection of karaj horticulture research division and also for studing the reaction of these cultivars against disease, the evaluation was performed by the USDA standard system for these cultivars in collection. In this study, although the most of cultivars had been infected on the natural conditions, but disease severity was significantly different between them. By the USDA system, pear cultivars were divided in the 2 classes. Percentage of pear cultivars in very susceptible and moderately susceptible classes were 81.4 and 18.6 respectively. Also for dividing the pear cultivars, beside of USDA method, SPSS software and the cluster analysis were also by the UPGMA method and cultivars were divided into 3 clusters, but it seems that the classification of cluster analysis did not conform with USDA system. Correlation of I.V.S in the artificial tests and the disease severity by natural infection was very significant (r= -0.83).

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Ascochyta blight caused by Ascochyta rabiei is one of the major diseases of chickpea (Cicer arientinum) in Iran. Many phytopathogenic microorganisms, incuding A .rabiei, attack their host plant by secreting pectic enzymes including polygalacturonase (PG) which causes modification of cell-wall structure, increasing accessibility of cell-wall components for degradation by other enzymes. Polygalacturonase is the major factor in the initiation of Ascochyta blight disease, therefore in this study, the enzyme was purified from a virulent isolate of A .rabiei (IK06). Fungi were cultured in PZ medium culture media were harvested and after dialysis used for purification. Purification was achieved by Carboxy Methyl Sepharose Fast Flow ion exchange column equilibrated to pH= 5.5. Zero to one molar NaCl gradient was used for elution of the proteins from the column. Determination of protein content and enzyme activity of each fraction showed that PG was eluted from the column in 0.3 to 0.4 M salt. The purity of the protein and the MW of the enzyme were determined using SDS-PAGE technique. The MW was found to be around 27 KDa. The activity of the purified protein was also evaluated using polyacrylamide gel containing pectin as substrate (zymogram gel). Optimum pH for the purified enzyme was 7.5.

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Fire blight caused by Erwinia amylovora, is a serious disease of pome fruits in many areas in the world which causes evaluative necrosis. Indeed, E. amylovora can invade the whole tree solely by internal progression through the host tissues. In this research, symptoms of necrotic shoots and exudates production on infected pear trees in different areas of Guilan province (Astaneh Ashrafieh, Lahijan and Kiashahr) were surveyed. Samples were taken from infected tissues of diseased trees. For isolation of bacterial causal agent, the infected tissues were crushed in pepton water, then 100µl of the extracts were cultured on Sucrose Nutrient Agar (SNA) and Luria Berthani (LB) containing Cyclohexamid antibiotic (50 µg / ml). The isolated bacteria were rod-shaped , gram negative and facultatively an-aerobic. The bacteria produced Levan on media including sucrose , but could not produce fluorescent pigments on King’s B medium. All strains made hypersensitive reaction on tobacco leaves. All isolates were oxidase , nitrate, urease and indole negative and were not able to rot potato tuber slices, produce H₂S and grow in 36 °C. The isolates could use citrate, acetoin, sorbitol and trehalose and their gelatin test was positive. Based on morphological, biochemical, physiological characteristics and production of a 937 bp with specific primer Ea1 and Ea2 in PCR method, the strains were identified as E. amylovora. This is the first report of the existence of this bacterium on pear fruit trees in Guilan province.

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To understand the role of relative humidity rate, host genotype, inoculation method and growth stage in epidemiology of bean common blight, two greenhouse experiments were carried out monitoring epiphytic population size of Xanthomonas axonopodis pv. phaseoli (Xap) and disease severity. The result showed significant differences among genotypes, inoculation methods and growth stages for epiphytic population size and sam effects except genotypes for disease severity. The epiphytic population size was significantly higher on spray inoculated Khomein cultivar of bean during flowering (R6). However, the relative humidity rates did not significantly affect population dynamics of epiphytic Xap and the disease severity. Two field experiments were also carried out to determine the effects of irrigation systems (furrow irrigation and overhead sprinkler irrigation), inoculation method, growth stage and their interactions on epiphytic population size of Xap and disease severity. The result showed that the epiphytic population size and disease severity were higher on spray inoculated plants irrigated with overhead sprinkler system during pods filling (R8). In this study, a significant positive correlation was found between epiphytic population size of Xap and bean common bacterial blight severity.

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Fusarium head blight (FHB) is one of the most destructive diseases of wheat causing significant reduction in grain yield and quality. Development of resistant varieties is an effective, economical and enviromentally safe way to control FHB disease. A major QTL (quantitative trait locus) for Fusarium head blight resistance, Qfhs.ndsu-3BS, derived from cv. Sumai 3, has been identified and verified by several research groups via molecular marker analysis. The resistant cv. Sumai 3 was crossed to susceptible cv. Falat, then three backcrosses were followed by one self-fertilization. Three simple sequence repeat (SSR) markers, Xgwm 389, Xgwm493, Xgwm533, were used for marker assisted selection (MAS) in BC1 and BC2 generations.The probability of linkage between markers and Qfhs.ndsu-3BS was calculated using a binomial probability function based on the assumption that a molecular marker at a specific distance from Qfhs.ndsu-3BS in the population would carry the donor-parent allel as a function of the distance between marker and QTL and the number of backcrosses/selfs used in deriving the population. Microsatelite locus Xgwm 493 was significantly associated with Qfhs.ndsu-3BS.