New species of Colletotrichum from wild Poaceae and Cyperaceae plants in Iran.

Twenty-two Colletotrichum strains were isolated from anthracnose symptoms or leaf spots on leaves of various wild Poaceae and Cyperaceae plants collected in three provinces of Iran and tentatively identified as belonging to the Graminicola species complex based on morphology. All strains were studied via a polyphasic approach combining colony characteristics, morphology and phylogeny inferred from multi-locus sequences, including the nuc rDNA ITS1-5.8S-ITS2 (ITS), partial sequences of the ß-tubulin (tub2), actin (act), manganese superoxide dismutase 2 (sod2), DNA lyase 2 (apn2) genes, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the intergenic spacer between the apn2 gene and the mat1 idiomorph (apn2/mat1). Six species were distinguished, including three new species, namely C. caspicum, C. persicum, and C. sacchari, and three previously described species, C. cereale, C. nicholsonii and C. sublineola. Comprehensive morphological descriptions and illustrations are provided for all species. Furthermore, this study provided new insights into the distribution and host range of known species.

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight.

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.

Pathogenicity of some Rhizoctonia solaniz isolates associated with root/collar rots on the cultivars of bean in greenhouse.

One hundred and eighteen isolates of Rhizoctonia solani were gathered from infected roots and hypocotyls of bean (Phaseolus vulgaris L.) grown in the fields of Tehran Province, Iran. Two isolates of the collected samples belonged to binucleate and 81 isolates to multinucleate of R. solani. The multinucleate isolates showed different anastomosis groups as AG-4 (subg. AG-4 HGI, AG-4HGII), AG-6 and AG-2. In greenhouse, pathogenicity tests carried out on bean cv. Naz in randomized design with 4 replications and each replication (pots) with 5 seeds of bean. Infection was done with seeds of wheat which were infected to the fungus with pasteurized soil. Results showed that the highest disease severity was caused by AG-4 (Rs21) isolates, whereas AG-4 (Rs74) isolates were weakly pathogenic with 90% and 21% infection, respectively. In this test the major pathogenic isolates belonged to AG-4 and they caused seed rot and damping-off of bean and AG-6 isolates were non-pathogenic. Five isolates of the fungus with major pathogenicity (Rs7, Rs18, Rs21, Rs62 and Rs71) selected and used for the reaction with different cultivars of bean. In this test, the cultivars and lines of bean (Pinto, red, white, green) studied in factorial experiment as randomized block design with 4 replications (pots). Results showed that none of the cultivars was completely resistant, however green bean cv. Sanry and pinto cv. Shad with number 4.8 disease severities had the highest susceptibility to seed rot and damping-off and red bean cv. Goli with 2.58 had the lowest susceptibility to the infection. Reaction of the cultivars and lines to the isolates of R. solani was significantly different at 1% level. Isolates of the fungus, Rs7, Rs21 with 84%, 90% pathogenicity was more virulent than the others.

Identification of anastomosis group of Rhizoctonia solani, the causal agent of seed rot and damping-off of bean in Iran.

Bean is one of the major crops in Iran. Seed rot and damping-off caused by Rhizoctonia solani is the most important disease of bean. In this research, infected roots and seedlings of beans were collected from different fields of Tehran Province. The samples were sterilized with 10% sodium hypochloride (5% stock) and incubated on PDA surface in petri-dishes. The purified fungi kept on filter paper and identified, pathogenicity test of R. solani was carried out on 2 cultivars of bean (red bean cv. Naz and white bean cv. Dehghan) and it determined. For identification of the anastomosis groups, the discs of cultured media with 5 mm. diameter of standard AG placed on one side of microscopic slides covered with water agar (2%) of 1 mm. thick and the isolates of the fungus on another side of slide about 2 cm away from each other. Experiment carried out in 4 replications. The cultures were incubated in 25 +/- 1 degrees C incubator for 24 hours, then the mycelial contact stained with lactophenol, cotton blue and hyphal anastomosis looked for under the light microscope with 10 x 40 and 10 x 100 magnifications. As a result, anastomosis groups: AG4, AG4HGII, AG2-2-2B and AG6 determined, frequency of these groups were 64, 18, 2, 16%, respectively. The group AG6 and subgroups AG4HGII and AG2-2-2B are introduced as new anastomosis groups on bean in Iran.

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity.

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.

Study on genetic diversity of Magnaporthe grisea using PCR and determination of the mating type alleles distribution in Mazandaran province, Iran.

The population structure of Magnaporthe grisea, the causal agent of the rice blast, was analyzed in Mazandaran province, using DNA fingerprinting based on RAPD-PCR by means of three primers including "I", "D" and "H". Total DNA of 47 isolates was extracted and amplified according to a specific PCR program. As a result, variable length fragments were generated. Each isolate was subjected to DNA fingerprinting and clonal lineages were determined. Phenetic analysis differentiated three distinct fingerprint lineages. In order to study on fertility status and distribution of the mating type idiomorphs (alleles), 72 monoconidial isolates from Mazandaran province were paired with four standard fertile hermaphrodite isolates. The mating type of 36 isolates was determined as Mat 1-1. The others (36 isolates) did not form any perithecia in pairing with standard isolates

Vegetative compatibility and pathogenecity of Verticillium dahliae kleb. isolates from olive in Iran.

Verticillium wilt caused by Verticillium dahliae is a serious problem of olive trees leading to significant reduction in yield. Verticillium wilt of olive trees was first recorded in Iran 1996 and confirm as due to Verticillium dahliae Kleb. 101 isolates of V. dahliae from olive trees at deferent locations in north provinces of Iran were assigned to vegetative compatibility groups (VCGS), using nitrate non-utilizing (Nit) mutants. A higher frequency of nit 1/nit 3 mutants (93%) was obtained compared with NitM (7%) with 10% of the isolates being assigned to VCG1 and 51% VCG4B and 19% VCG2A. 20% of isolates could not be classified in standard isolates. The pathogenecity of 15 randomly selected isolates (5 of each VCG) was tested on olive seedling (cv. Zard) and eggplant. The VCGs isolates were similarly aggressive on olive. However, VCG1 isolates were more aggressive on eggplant cv. Local than the VCG2A and VCG4B isolates as indicated by a higher colonization index. The pathogenecity tests of the pathogen on test plants (cotton cv. 'sahel', eggplant cv. 'local' and tomato cv. 'ps') show all isolates category in 2 pathogenecity groups defoliate and non-defoliate (with severe and mild subgroups). The morphology of V. dahliae isolates on C'zapeck's agar and water agar medium were different especially for microsclerotia appearance time in culture and their morphology.

Study on DNA diversity of Iranian populations of Erysiphe betae causal agent of sugar beet powdery mildew.

107 samples of E. betae were collected on infected leaves from all over Iranian beet cultivation areas. Their choosing were based on geographical and host origin(sugar beet, red beet, fodder beet and wild beet). 30 isolates were single colonized and grown on sugar beet susceptible genotype 7233. 107 specimens were analyzed by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8s DNA which previously amplified by the polymerase chain reaction (PCR) with 2 universal primers, ITS1 and ITS4. PCR product was affected by 9 different restriction enzymes. PCR product was a 645 bp band for all of the isolates. 3 restriction enzymes; CfoI, MspI and HaeIII could cut this fragment into smaller bands, but electrophoretic patterns were identical for all of the isolates. 30 single colonized isolates were used in RAPD experiments. In RAPD-PCR experiment genetic diversity was investigated with 30 isolates from different parts of the country. 59 random primers were used and then 21 primers that displayed good consistency and reproducibility were selected. Most of the primers revealed identical patterns between 3 to 14 bands. 5 primers that showed more polymorphism were selected to analyze 30 isolates. For these 5 primers 61 distinct bands were obtained which 62% of these bands were polymorphic. Results indicated that there is no relationship between cluster grouping and geographical origin and the isolates showed a high similarity.

Olive verticillium wilt or dieback of olive in Iran.

During 2000--03, different areas in Zanjan, Golestan and Khorasan provinces were surveyed for the presence of olive dieback. Olive branches, leaves and roots showing typical symptoms and soil around the roots were collected for further study. Samples were surface-sterilized with sodium hypochlorite or ethanol and then cultured on PDA and Czapek media. Soil samples were diluted in ethanol-agar for fungal isolation and purification. Morphological characteristics of the fungal mycelium particularly phialide and spores identified the causal agent to be the soil-borne pathogen, Verticillium dahliae. The disease was present in all olive growing regions but it was severe in temperate and relatively humid regions such as Gorgan. Infection index of the disease varied between 5 to 30% with an average of 11.89+/-1.12 among various orchards in this area. The newly established orchards showed more infection than the older ones. A significant difference in disease incidence and severity were observed among olive cultivars of Michen, Roughani, Zard and Koronakei. The latter cultivar had the least amount of infection. Strains of V. dahliae isolated from olive trees had different morphological and pathogenicity characteristics. These strains had different growth rates in response to the optimum temperature of 20 or 25 degrees C. The number of fungal propagules per gram of air-dried soil ranged from 2 to 32 with an average number of 13.42+/-0.50. Regarding the number of propagules of V. dahliae in the soil and susceptibility of cultivars in the newly established orchards, it seems necessary to take serious control measures to prevent disease spread.

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran.

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.

The assessment of the rice cultivars/lines resistance to blast disease in Mazandaran province, Iran.

Blast, caused by Magnaporthe grisea, is one of the most important diseases in rice production regions of the world including Iran. To determine progress of rice blast disease on the selective cultivars and lines also to assay some components of partial resistance, a set of Iranian rice cultivars (Local and breeding) along with near-isogenic lines (NILs) and breeding lines from International Rice Research Institute (IRRI) were tested with some field races of the fungus in blast nursery and five of selective races in greenhouse. These experiments were conducted in a Randomized complete Block Design (RCBD) with three replications (except greenhouse experiment on the leaves). Traits in this study consisted of Infection Neck Number (INN), Neck Lesion Size (NLS), Infection Type (IT), percent Diseased Leaf Area (DLA) and Area Under Disease Progress Curve (AUDPC); also IT, Sporulation Lesion Number (SLN), Sporulating Region Diameter (SRD) and percent DLA were measured in leaf blast in greenhouse (one replication). The Iranian local cultivars and NILs i.e. Co-39 and C104-PKT located as susceptible group for AUDPC, IT, INN and NLS. Iranian breeding cultivars, breeding lines from IRRI and NILs (except Co-39 and C104-PKT) were resistant or indicated hypersensivity reaction (HR). Some cultivars (Fujiminori, Onda, and Hassan Saraii) were semi susceptible to leaf blast in nursery. The main point is correlation in 1% (a = 0.0001) between the traits in greenhouse and blast nursery. Neck node infection of Haraz cultivar in greenhouse experiment to IA-89 race is very important, because Haraz is a resistant cultivar to blast disease in Iran.