فصلنامه بیماریهای گیاهی
سال چهل و نهم شماره 4 (زمستان 1392)

This study was conducted on the interaction between Verticillium dahliae, the causal agent of vascular wilt of pistachio trees, and Acremonium kiliense, an endophyte fungus with biological control effects. Nine-month-old seedlings of three pistachio cultivars including Sarakhs, Badami-Rize-Zarand and Ghazvini were used by root dip method in conidial suspension (106 ml-1) and transferred to pots containing autoclaved soil. After one month, the inoculated seedlings were transferred to soil containing 40 microsclerotia/g soil of V. dahliae. The trial was performed as a two-factor factorial experiment with 3 replications for each combination, the factors being cultivars and fungal treatments at 3 and 4 levels, respectively, in a completely randomized design. The results showed an increase in the dry weight of roots and shoots in trees inoculated with A. kiliense plus V. dahliae compared to trees inoculated only with V. dahliae. Interaction of A. kiliense and V. dahliae decreased the percentage of shoots and roots infection compare to treatments of V. dahliae alone. The percentage of V. dahliae isolation from different treatments showed that cultivars inoculated with A. kiliense plus V. dahliae had a lower colonization by V. dahliae than those inoculated only with V. dahliae. The results of statistical grouping revealed that shoot dry weight in all inoculated cultivars with A. kiliense, A. kiliense plus V. dahliae, and control belonged to one group, whereas cultivars inoculated only with V. dahliae constituted a separate group. The results of the Verticillium isolation from tissue showed that the isolation rate of V. dahliae in interaction between A. kiliense-V.dahliae, compared to V. dahliae alone, decreased more than 50 percent in all cultivars.

Wheat dwarf virus (WDV) is one of the main factors causing stunting and yellowing diseases of cereals. Two strains of WDV have been reported from different parts of Iran. The barley strain (WDV-B) is only detected from naturally infected barley plants, while the wheat strain (WDV-W) infects both wheat and barley plants. Previous results regarding distribution of WDV strains have revealed the dominance of WDV-W compared to WDV-B in cereal fields of Iran. To investigate the taxonomic position of the Iranian WDV strains, the complete DNA genome of a wheat isolate of WDV-W from Shahrekord (WDV-Whe[Iran:Sh:Whe]) and of a barley isolate of WDV-W from Bavanat (WDV-Whe[Iran:Ba:Bar], with 2750 and 2749 nucleotides, respectively, were sequenced. Phylogenetic analysis of nucleotide sequences of the genome, revealed two main clusters designated WDV- W and WDV-B. Cluster WDV-W is composed of two Iranian isolates including WDV-Whe[Iran:Sh:Whe], WDV-Whe[Iran:Ba:Bar] and nine non-Iranian wheat isolates of WDW (WDV-W). An Iranian barley isolate of WDV (WDV-Bar[IR:Bar]) and seven exotic barley isolates of WDV formed the second cluster (WDV-B). Sequence comparison of different genomic regions of the two Iranian WDV-W isolates showed that the replication-associated protein was the most conserved while the movement protein and the large intergenic region were the most divergent regions.

In order to identify nematodes associated with orchards and fields of Kahak and Markazi regions in Qom Province, 50 soil and root samples were collected from different localities, during spring and autumn of 2010. The samples were washed and nematodes were extracted by centrifugal floatation technique. They were fixed and transferred to glycerin according to the de Grisse method (1969). Permanent slides were prepared from the extracted nematodes and some cross sections were made from different parts of the body. Morphological and morphometrical characters specimens were studied by light microscope attached to drawing tube. In result 16 species belonging to 12 genera of Tylenchina were identified. From these species Aphelenchoides singhi, Boleodorus volutus and Prothallonema macrocellum are reported and described for the first time from Iran and Aphelenchoides singhi is reported for the second time in the world..

Among begomoviruses infecting tomato, Tomato leaf curl Palampur virus-IR (ToLCPMV-IR) is the first bipartite begomovirus reported in Iran. To demonstrate infectivity of the bipartite genome and identify the experimental host range of the ToLCPMV-IR, dimer construct for each genomic component A and B was designed and constructed. The resulting clones were separately ligated to binary vector pGreen0029 and used simultaneously with pSoup (helper plasmid) to transform Agrobacterium tumefaciens strain C58. Agroinoculation with the cloned DNA of the ToLCPMV-IR genome resulted in the efficient infection of several experimental hosts such as cucumber, squash, tomato and three tobacco species but not watermelon. The virus was transmitted from agroinoculated plants to healthy squash seedlings by whitefly Bemisia tabaci, the natural vector of ToLCPMV-IR. Based on the results of this study, we demonstrate infectivity of the bipartite cloned genome of ToLCPMV-IR. The constructed infectious clone can be used for evaluation of the reaction of host plant cultivars and analysis the genes function.

During spring and summer of 2010 and 2012, several surveys were conducted in different vineyards in Kohgiluyeh & Boyer-ahmad (southwestern Iran) and Kerman (southeastern Iran) provinces, with the aim of isolating and identifying fungi associated with grapevine decline diseases. The samples collected comprised diseased grapevines showing yellowing, defoliation, reduced growth, wilting, interveinal chlorosis, internal black spots and wood discoloration in cross section, as well as woody vine debris taken from the vineyards floor. Isolations were made from symptomatic wood tissues and pycnidia on the bark of pruning debris on malt extract agar supplemented with 1 mg ml-1 streptomycin sulphate (MEAS) and potato dextrose agar (PDA) media. Based on morphological and molecular characteristics four species: Phaeoacremonium tuscanum, Diplodia seriata, Botryosphaeria dothidea and Neofusicoccum parvum were isolated and identified from samples collected. In this regards, D. seriata and B. dothidea were recovered from diseased grapevines and pruning debris while N. parvum was obtained only from pycnidia formed on the bark of grapevine pruning debris. Pm. tuscanum was isolated from grapevines showing yellowing, defoliation, small chlorotic leaves, internal black spots and brown to black streaking in cross section. Pathogenicity tests were conducted on green shoot cuttings of 'Askari' cultivar under greenhouse conditions. Based on the results of pathogenicity tests, all tested species were pathogenic and caused significant wood discoloration on inoculated cuttings 25 days after inoculation. The fungi were re-isolated from the margin of the lesions, completing Koch’s postulates. Based on our knowledge, this is the first report of Pm. tuscanum from grapevine in Iran. This is also the first report of D. seriata, N. parvum and B. dothidea from pruning debris on the vineyard floors in this country.

Fusarium damping off caused by Fusarium oxysporum f.sp. sesami is one of the most important diseases of sesame, in all areas of sesame production in the world. In this investigation, resistance of 20 sesame germplasms to Fusarium damping off was assayed in greenhouse. Fifteen isolates of the causal agent were isolated from sesame in Yazd region and one isolate with the highest pathogenicity potential selected. Special test showed that the isolate was Fusarium oxysporum f.sp. sesami (FOS). Twenty sesame germplasms were assayed by disease severity index (0-2) and infection percent index (1-5) for their reaction to FOS. Statistical analysis of data showed that there was a considerable difference at about 1% level among the investigated germplasms from the perspective of resistance to disorder facror. By using the achieved findings determined that local Asfige Bahabad germplasm as resistant germplasm and local Kahnouj germplasm as susceptible germplasm. In the next step of study, PAL activity was assayed in resistant and susceptible germplasms 2, 4, 6, 8, 10 and 12 days after inoculation.PAL enzyme activity in resistant germplasm increased rapidly and reached it’s acme in the 4th day after inoculation and then decreased, increased to 12th day again. But this amount was lower as 4th day. In susceptible germplasm PAL activity increased slowly. This investigation showed that the increase of PAL activity can have probable role in the induction of resistance.

Monitoring of the behavior of biocontrol agents in situ is a powerful tool for successful management of diseases. In this research a Trichoderma isolate was identified as T. harzianum by the analysis of rDNA-ITS regions using the TrichOKEY and TrichoBLAST sequence identification programs. For gfp transformation the maximum protoplast yield (5 × 108/ml) was obtained using the optimized conditions of protoplast isolation. The highest transformation efficiency (96%) was obtained with 10 µg DNA and 30 µl protoplast (5 × 108/ml). To obtain a genetically marked Trichoderma that can be used as a biomonitor, T. harzianum was cotransformed with p3SR2 and pDL2 plasmids containing amdS and gfp genes, respectively. Transformation of gfp gene was confirmed by PCR pattern. The morphology and viability of selected cotransformant (T. harzianum T8-7MK) was similar to that of the wild type. The biocontrol activity of transformant was confirmed by Dual culture with Rhizoctonia solani and microscopic analysis. The monitoring of gfp tagged T. harzianum was followed in agricultural soil (in 60 days after inoculation). Also the colonization of T. harzianum T8-7MK was monitored visually on R. solani sclerotia by scanning electron microscopy. The treated sclerotia showed no detectable growth on PDA after six days, while the untreated sclerotia were able to grow after two days on the same medium. These observations confirmed the biomonitor and biocontrol activity of T. harzianum T8-7MK transformant.

Cercosporoid fungi including the genus Cercospora and other morphologically similar genera are important pathogens causing leaf spots on a wide range of host plants. In a taxonomic study on cercosporoid fungi, plants with leaf spot symptoms from different localities of Mazandaran province were collected and examined during spring-autumn 2011-12. As the result, Cercospora hostae (on Hosta albomarginata), Pseudocercospora cydoniae (on Chaenomeles japonica) and Stigmina palmivora (on Washingtonia robusta and Phoenix canariensis) are reported here as new records for Iran. Hibiscus syriacus is a new host for Cercospora althaeina. Furthermore, Cercospora apii s. lat. is identified on 13 host plants including 6 new hosts in the world and 5 new hosts in Iran.

Absidia is a genus in the family Absidiaceae in the order Mucorales some species of which are causative agents of mucormycosis in humans and animals. To study phylogenetics and population biology of the genus and its species, some some soil samples were collected from agricultural areas of Mazandaran. Following isolation and purification of isolate an Rose-bengal containing medium, 20 isolates of Absidia were obtained. DNA was extracted from using a CTAB method and subjected to PCR using the primers ITS4 and ITS5 and the amplified fragments were sequenced. The sequences were analyzed by Chromas Pro and MEGA5 softwares and compared with those deposited in GenBnak. Dendrograms derived from the similarity of nucleotide sequences separated Absidia repens isolates, which formed a distinct calde, from the rest of the species.

The effect of various temperatures on the infection of sugar beet plants by Iranian isolate of Beet severe curly top virus (BSCTV-IR) was evaluated. Presence of BSCTV-IR in agroinoculated plants was assayed at 7, 14 and 21 days post-inoculation (dpi) using PCR. Based on the infectivity assay, the optimum temperature for BSCTV-IR infection was 25C at which the mean period between inoculation and the first virus detection was the shortest and the virus concentration was the highest. No virus was detected in plants incubated at 35C until 21 dpi. To evaluate the effect of temperature on recovery of BSCTV-IR-infected plants, symptomatic plants were incubated at the aforementioned temperatures. The first new leaf showing recovery phenotype emerged 7 dpi at 35 ºC. This period for plants incubated at 30, 25-30, 25 and 20ºC was 12, 18, 25 and 28 days post-incubation, respectively. Thus, recovery occurred faster as the temperature raised. Re-inoculation of recovered plants with the same virus induced no symptoms in newly emerging leaves, indicating the stability of the phenotype of recovered plants against re-infection by the same virus. Quantitative analysis using real-time PCR showed significant decline in viral DNA in either recovered or new leaves emerged after re-inoculation compared to symptomatic leaves.

This study was conducted in 2010-2011 to evaluate the effects of herbicides trifluralin, ethalfluralin, alachlor and metribuzin on the incidence of damping off disease caused by Rhizoctonia solani on soybean varieties Williams, Mill 82 and Clark in sterile or non-sterile soil. The pathogen was grown on PDA and incorporated into the soil of each pod up to four cm depth. The incidence of the disease in herbicide-treated soil was compared with those of untreated soil which was pre-inoculated with the pathogen as control. In these experiments, Clark showed higher disease incidence than other varieties. Also the disease incidence was higher in sterile soil than in non-sterile soil. Compared with the untreated control, only trifluralin, significantly increased the disease incidence.

Canker symptoms similar to those produced by Pseudomonas syringae pv. Syringae were observed on peach (Prunus persica) and nectarine (Prunus persicae var. nusipersica) trees in several fruit tree growing areas in Khorasan in 2011-2012.Symptomatic stem tissues were thoroughly washed under running tap water, surface disinfestated with sodium hypochlorite (2 min. in 0.5 %, ai, hypochlorite), washed in three changes of sterile distilled water (SDW) and the bark tissues were stripped-off and chopped in 1-2 ml of SDW with a sterile scalpel. Drops of the suspensions, following 10-20 min standing at room temperature, were streaked onto plates of sucrose nutrient agar (SNA) and the plates were incubated at 26-28 °C. The predominant isolates recovered had cream to pinkish, circular, glistening, pasty and convex colonies with entire margins. The isolates did not produce fluorescent pigments on medium B of King. A number of isolates induced a hypersensitive(HR)-like reaction in geranium (Pelargonium × hortorum) and in tobacco (Nicotiana tabacum) 4-5 and 11 days after injection of somewhat turbid suspensions, respectively. The necrotic areas or spots formed on tobacco were surrounded by halos and tended to enlarge, albeit, slowly. DNA of a representative from each group of isolates, differentiated by their colony morphology on SNA, was extracted and the ITS region of the rDNA operon was amplified in PCR and sequenced. Based on the identity of the nucleotide sequence of this region with those deposited in GenBank, the two colony types that were proved to be pathogenic to peach and induced a Hr-like reaction in tobacco and geranium were found to belong to the yeast species Cryptococcus adeliensis and C. magnus. Koch’s postulates were fulfilled with representative of both species on peach budlings(Borhani et al. 2012. 11 th European Conference on Fungal Genetics, Philipps-University, Marburg, Germanyand Borhani et al. 2012. 13 th International Congress on Yeasts, Univ. Wisconsin, Madison, USA).A number of isolates displayed characteristics distinct from those of the two Cryptococcus species described. Colonies of the present isolates were glistening, pasty, milky white in color and very low convex. Blast analysis of the ITS sequence of a representative (NCBI # JQ039905) of this group of isolates, which produced a Hr-like reaction in geranium but appeared to be avirulent on peach in greenhouse tests, showed 100% identity with those of Meyerozyma guilliermondi isolates present in GenBank. The fatty acid methyl ester (FAME) compositions of a representative isolate and a reference of strain Meyerozyma guilliermondi,CBS # 8417, were determined by gas chromatography. The similarity of the two isolates was verified further by the identity of the types and the percentages of fatty acids. C18: 1 was determined to be the predominant fatty acid (40% of the total fatty acid compositions) in both isolates. The role of this third group of yeast isolates, if any, in the stem canker complex, could not be established in the preliminary pathogenicity trials carried out in the present study and remains to be determined in the future. Induction of hypersensitive reaction on geranium may be considered as the potential of this yeast species in causing disease in one or more plant species.

Tristeza caused by Citrus tristeza virus (CTV), is the most important viral disease of citrus worldwide. Serious damages of the disease have been reported from many citrus growing areas of the world. CTV particles are restricted to phloem and are transmitted by aphids in a semipersistent manner. CTV was introduced from Japan to Mahdasht orchards in Mazadaran province in 1969. Its natural spread with the vector Aphis gossipii was shown in the infected areas (Rahimian et al., 2000). Following the transport of virus-infected scions or plants from the east of Mazandaran (infected area) to the western part of the province (Tarviji et al., 2012), the risk of transmission was considered by a similar way in the nurseries of Guilan province. Preliminary survey of the disease was carried out in November 2012. Five of 20 Unshiu (Citrus unshiu) and Thomson navel (C.sinencis) plants on Citrange rootstock were found infected by serological direct tissue blot immunoassay using a commercial antiserum (Bioreba, Switzerland). Infection of the plants was confirmed by a two-step RT-PCR with T36cp+/T36cp- primers after nucleic acid extraction with Rneasy Plant Mini Kit (Qiagen). RT and PCR was carried out respectively according to manufacture protocols of Revert Aid and PCR Master Mix (Fermentas).