فصلنامه بیماریهای گیاهی
سال چهل و هفتم شماره 1 (بهار 1390)

Verticillium wilt incited by Verticillium dahliae Kleb. is one of the destructive diseases of cotton in Golestan Province. In order to determine the effect of disease on the quantitative and qualitative characters of cotton yield and the phenotypic and genotypic correlation between characters of cotton plant yield and its tolerance to Verticillium wilt, 1500 plants were selected and grouped into five categories based on the rate of disease severity. In each group, 100 plants were selected and evaluated for rate of seed germination, quantitative and qualitative characters of yield, and weight of 1000 seeds. Characters were evaluated using SAS software based on a completely randomized block design with four or five replications. Fitfty cotton cultivars of Gossypium hirsutum with different characteristics were planted in a completely randomized block design with four replications in infected and non- infected soil. Then the disease indexes of yield, boll weight, early maturity, sympodial length, monopodial number, monopodial length of each cultivar were measured. The correlation between characters were then evaluated using SAS and Gen.cor software. The results showed Verticillium wilt reduces the rate of seed germination, boll weight, yield, ginning turnout, weight of 1000 seeds, plant height, number of leaves, number of bolls, number of nodes, oil percentage in wet seed, elongation, fiber fineness, length of fiber, fiber strength, fiber uniformity and percentage of oil in dried seed. The results also showed positive and significant genetic correlation between disease index and sympodial length (0.34), monopodial number (0.63) and monopodial length (0.69)., and negative and significant genetic correlation between disease index and boll weight and total yield. Positive and significant phenotypic correlation was also found between disease index and monopodial (0.31). There were also negative and significant correlations between the disease index, sympodial length and boll weight of (-0.21 and -0.18).The infected plants had less monopodial numbers and boll weight than healthy plants. Therefore, tolerant cultivars had more monopodial and sympodial branches and longer sympodial length than sensitive cultivars.

Many isolates of Trichoderma spp. and Bacillus subtilis are natural biological agents of plant pathogenic fungi and bacteria. Trichoderma spp. and B. subtilis isolates were separated from the rhizosphere of infected and non-infected rapeseed plants. Phoma lingam, the causal agent of rapeseed black leg, was isolated from different tissues of infected plants, and infested soil. Dual cultures were used to examine the antagonistic effects of Trichoderma and B. subtilis isolates on P. lingam. In the greenhouse, the antagonistic effects of the isolates were tested through treating rapeseed seeds and rapeseed above-ground parts with the isolates. Totally, 20 isolates of P. lingam, 15 isolates of Trichoderma species and 17 isolates of B. subtilis were separated. Bacterial isolates BE3, B31, B66, B67, B68, B69, and B70, and Trichoderma isolates Tr.2901, Tr.2903, Tr.2904, Tr.2910, and Tr.2913 showed antagonistic effects on the pathogen. The greenhouse results revealed that in the seed treatment experiment, the bacterial isolates B70 and B67, and Trichoderma isolates Tr.2910 and Tr.2901 decreased the rapeseed blackleg to 43.3, 80, 16.6 and 56.6℅, respectively. Treating the above-ground parts with bacterial isolates did not reduce rapeseed blackleg at all. Treating above-ground parts by Tr.2910 and Tr.2901 isolates of Trichoderma decreased the disease to 60 and 79.6℅, respectively. Trichoderma isolates belonged to several species where the differentiation of the isolates from species was impossible based on the morphological characteristics. The two effective bacterial isolates were related to B. subtilis.

Potato black dot disease, caused by Colletotrichum coccodes, is a common disease in many potato planting areas in Iran. This pathogen causes yield loss in field and tuber rot in storage conditions. Genetic diversity and pathogenicity of C. coccodes was unknown in Iran. In this study, C. coccodes was isolated from potato plants with black dot symptoms from Ardebil, Isfahan and Hamadan provinces. Amongst 108 isolates, 48 isolates were studied for vegetative compatibility groups and aggressiveness. Ten VCGs including four multi-member groups (IRN-VCG1, IRN-VCG2, IRN-VCG3 and IRN-VCG4) as well as six single-member groups were detected. Isolates of Ardebil were grouped in IRN-VCG1and IRN-VCG2 with 50 and 12.5% frequency and other isolates were single-member. Three groups of IRN-VCG1 (55%), IRN-VCG2 (13%) and IRN-VCG3 (13%) were constituted for Hamadan province isolates. Isfahan isolates were grouped as IRN-VCG1, IRN-VCG2 and IRN-VCG4 at 25, 19 and 12 percentage respectively, and 44% of isolates were not assigned to any of the four VCGs. The aggressiveness of the isolates were investigated using potato tuber decay assessment and root inoculation method. Most of isolates were able to decay potato tubers, while all isolates colonized the roots and were considered as aggressive isolates. Comparison of root and tuber inoculation methods showed no relationship between tuber rot severity and microsclerotia spreading in roots and stems. There was no correlation between VCGs and virulence while some correlation was detected for VCGs and geographical origin of the isolates. This study demonstrated that there is a significant VCG diversity in C. coccodes.

Several potyvirids infecting gramineous plants have been reported from Iran. High level of variation and similarities among these viruses has prompted us to compare them and precisely differentiate them. To this end, Iranian Johnson grass mosaic virus (IJMV) from Shiraz and Khuzistan, Bermuda grass mosaic virus (BgMV) from Bermuda grass in Shiraz, sugarcane mosaic virus (SCMV) from sugarcane in Khuzistan, Bermuda grass southern mosaic virus (BgSMV) from Bermuda grass in Jiroft and Safiabad (Khuzistan), and maize dwarf mosaic virus (MDMV) from maize in Dasht-Naz (Sari) were collected, preliminary tested serologically and compared by host range, transmission, serological and molecular studies. Serological tests of agar gel diffusion (AGD), ELISA and western blot analysis were conducted using MDMV-G (German isolate), MDMV-Ir, BgSMV, IJMV and BgMV antisera or immunoglobulins. The results showed that BgSMV has a close serological relationship with MDMV but in AGD they were differentiated from each other by spur formation. The molecular weight of the coat protein of BgSMV was higher than that of MDMV. IJMV also differed in the size of coat protein. Serological comparison revealed that IJMV, SCMV, BgSMV and MDMV showed a weak serological relationship with each other, but not with BgMV. In host range studies, all isolates, but BgMV, infected all cultivars of sorghum and maize. Pearl millet was infected only by MDMV and BgSMV isolates. BgMV only infected goose grass. Among viruses, only MDMV-Ir and IJMV were transmissible to Johnson grass. None of the virus isolates could infect wheat, barley and rye. Both IJMV and MDMV-Ir were transmitted to sorghum by Rhopalosiphum padi, R. maidis and Schizaphis graminum. Therefore, MDMV could not be differentiated from IJMV by these vectors. BgSMV was not transmitted by R. maidis. BgMV was distinct from other potyviruses as it was transmissible by Sipha elegans and Hysteroneura sp. For molecular studies a portion of the gene corresponding to CP core region was amplified by RT-PCR, cloned and sequenced. Multiple alignment was performed with some other cereal potyviruses and phylogenetic tree was depicted. Results showed that IJMV isolates were grouped with Zea mosaic virus (ZeMV) in the same clade. MDMV from Iran was grouped with European isolates of this virus. Another clade included BgSMV isolates and isolates of MDMV, although BgSMV isolates were grouped in a separate branch. Iranian isolates of SCMV were close to American and Australian isolates of this virus. In summary, five distinct potyvirid species, not to mention members of Tritimovirus and reed canary grass mosaic virus which infects Phalaris arundinacea, were detected on gramineous species in Iran. IJMV and MDMV infect Johnson grass and maize. BgSMV infects Bermuda grass and is restricted to southern warm regions of Iran. SCMV was only isolated from sugarcane in Iran. BgMV, a completely distinct virus, was detected on Bermuda grass and is widespread in temperate and cold regions of Iran.

Barley and cereal yellow dwarf are the most important cereal diseases causing serious damages worldwide. Limited number of resistance and tolerance genes, their inefficiency and their complex inheritance justifies the adoption of the pathogen derived resistance approaches. In the present study, three types of constructs including sense, antisense and intron hairpin-loop (ihp-loop) were prepared from a sequence of BYDV-PAV polymerase gene and cloned into pWBVec8 binary vector. Agrobacterium-mediated transformation of mature embryos of bread wheat Alvand was carried out using the three types of constructs in the optimized culture media supplemented with hygromycin as a selective marker. The transformed explants were evaluated by ELISA, polymerase chain reaction, and dot blot assay using a nonradioactive probe. Artificial inoculation of plants was carried out employing BYDV-PAV viruliferous aphids. The average rates of transformation were evaluated as 1.8, 1.3 and zero percent for ihp-loop, sense and antisense constructs, respectively, based on ELISA values. Transformed plants remained healthy while control plants developed yellow dwarf symptoms after artificial inoculation of BYDV-PAV.

During 2007-2009, 156 soil samples were collected from the most important Pistachio orchards of Kerman province. Nematodes were extracted from 250g soil by sieving and centrifuge method and Longidoridae nematodes were counted. Nematodes were fixed with 4% formalin and processed by De Grisse method, and permanent slides were prepared. Using a light microscope equipped with a drawing tube, morphological and anatomical characteristics of nematodes were studied, measurements were carried out and the species of nematodes were identified. Species were compared with the original descriptions. Longidorus africanus, with maximum population and distribution was dominant species in many soil samples, while Xiphinema vuittenezi, X. pachtaicum with medium population were found in some samples, and X. index with minimum population was also identified. X. oxycaudatum is for the first time reported from Kerman province.

In this study, 99 Rhizoctonia solani isolats were isolated from some pistachio nurseries in Kerman province. Fungus was isolated from infected plant tissues and soils. Isolates were identified according to the morphological characters of the hyphae, cellular nuclei number of young hyphae, and their anastomosis groups. Results revealed that 96 isolates were multinucleate (MNR) and 3 of them binucleate (BNR). MNR isolates belonged to AG-4, and BNR isolates to AG B-I. Pathogenicity of the isolates was carried out on pistachio plants and the MNRs were pathogenic on pistachio but their virulence levels were significantly different. None of BNR isoltes were pathogenic on pistachio. The applicability of biological control as a method for combating disease was investigated in lab and greenhouse experiments using BNR isolates. In dual culture tests, no hyphal lysis was observed and the only interaction was coiling of the bc agent hyphae around the R. solani hyphae. In greenhouse experiments, coinoculation of the plants by BNR and R. solani did not control the disease. On the other hand, when plants were inoculated by BNR, two weeks before inoculation with R. solani, the disease was reduced considerably.

The study on recently collected specimens of Populus x euramericana infected by a Melmpsora species revealed that the specimens are different from other Melampsora species recorded on Populus in Iran. Uredinia and telia were found on the infected plant. Uredinia hypophyllous, scattered, pulverulent, and round often covered the whole leaf surface; urediniospores were mostly obovoid, 31-43 x 15-20 µm, their wall echinulate, smooth at apex, 1.5-2 µm thick at apex, up to 6 µm thick at sides; and paraphyses were clavate, their wall up to 13 µm thick at the apex. Telia was black, amphigenous but mostly epiphyllous, sub epidermal; teliospores adhered laterally, 40-64 x 8-12 µm, wall 1 µm thick, apically thickened up to 2.5 µm. Fungal morphological features matched features characteristic of Melampsora larici-populina Kleb. This is the first report of a rust disease attacking leaves of Populus x euramericana in Iran. Melampsora larici-populina is also newly reported for the Iranian Rust mycobiota.

Rhizoctonia solani Kühn AG 1-IA [teleomorph = Thanatephorus cucumeris (Frank) Donk] is the causal agent of rice sheath blight, which is one of the most serious diseases of rice worldwide in both tropical and temperate climates. The disease occurs throughout rice production areas in southern coast of Caspian Sea, the major rice growing region in Iran, and is considered as the most important rice disease on susceptible semi-dwarf, high-yielding cultivars. The perfect stage of the pathogen has been frequently observed on rice as well as on alternative hosts in several countries but up to now there has been no report from Iran. On August 2008, during a warm and humid season, rice plants (cv. Neda) from a hill with severe sheath blight symptoms bearing a superficial powdery growth spreading over tillers was observed at the beginning of ripening stage in a field near Amol, Mazandaran. Hymenia were effuse (Fig. 1A), cream to grayish white in color and composed of an arachnoid network of hyphae (7-11.5 µm in diameter) from which clusters of basidia were arisen. Light microscopic analysis revealed barrel-shaped basidia, subclavate, some cylindrical, slightly wider than the supporting hyphae, 11-16 × 7-9 µm (Fig. 1B). Sterigmata were long, stout, tapered, usually four (rarely two or three). Basidiospores were hyaline, oblong to ellipsoid, some obovate, with a prominent apiculus, 7.5-9 × 4-6 μm (Fig. 1C). On the basis of above characteristics, the Basidiomycetous fungus on rice was identified as T. cucumeris. This is the first report of naturally occurring T. cucumeris on rice plants in Iran. Single basidiospores germinating on water-agar were transferred to potato dextrose agar plates and incubated at 27°C. The fungal colonies was 2.1 and 4.0 cm in diameter after five and nine days of incubation, respectively. Colony was first snow white then became cream to beige. Hyphae resembled R. solani’s, especially in nearly right-angled side branches with basal constriction. For the pathogenicity test, rice seedlings (cv. Neda) were transferred to plastic pots containing rice field soil. Inoculation was done by placing a portion of the actively growing culture (derived from a single basidiospore) between leaf and stem of 40 day old rice plants. The glasshouse temperature varied from 27-32 °C and relative humidity from 90  5%. Plants were inspected for disease symptoms, one, two and three weeks after inoculation, but no lesion was observed. It is plausible that sexual basidiospores produced as a result of heterothallic mating might constitute an important aspect of the fungal life history contributing to the genetic diversity of field populations of R. solani AG-1 IA anywhere rice is cropped, including Iran.

Huanglongbing is an important disease of citrus and has been regarded as a quarantine diseases in Iran. The disease has been recently reported from some parts of Sistan-Baluchistan and Hormozgan(Alizadeh 2009). The causal agent of the disease in Iran is the bacterium Candidatus Liberibacter asiaticus vectored by citrus psylla. Proximity of Kerman province with Sistan-Baluchistan and Hormozgan provinces, observation of huanglongbing-like symptoms and difficulties of symptomatic diagnosis of the disease prompted us to use a molecular method for identification in Orzooiyeh District. In September, 2010, in an attempt to trace the disease, samples were collected from 120 suspected trees. Total DNA was extracted from vein of the samples using DNA Extraction Kit (Fermentas). DNA samples were tested by PCR using two pairs of primers. Primers A2 and J5 amplified a 703 bp segment of ribosomal protein genes in Asian species while the primers OI1 and OI2C amplified a 1160 bp segment of 16s rDNA. The amplified DNAs (PCR products) were analysed on %1 agarose gel stained with ethidium bromide. For both primer pairs, the expected band of PCR product was observed in one sample. Presence of 703 bp band in product of PCR test using A2 and J5 primers, and also result of enzymatic digestion of PCR product related to OI1 and OI2C primers by enzyme XbaI and observing two bands of about 520bp and 640bp confirmed the presence of Candidatus Liberibacter asiaticus. PCR products were sequenced for further investigation. The infected tree was a four years old tangerine, Kara variety, in Qader-abad, Orzooiyeh, exhibiting symptoms such as dark and bright patches of yellow, bright green and dark green without any definite margin on the surface which may also be caused by other agents. Moreover, other symptoms such as pox-like irregular distortions on the fruit, tiny, shrunken, hollow, sterile and dark brown seeds and asymmetry of the fruit were observed. This is the first report of hunglongbing in Kerman province (Orzooiyeh). The infected tree was eradicated according to the instruction received from Plant Protection Organization.