فصلنامه بیماریهای گیاهی
سال چهل و ششم شماره 3 (پاییز 1389)

Leaf rust of wheat caused by Puccinia triticina Eriksson occurs annually in different parts of Iran. Samples of Puccinia triticina were collected from rust-infected wheat leaves from different regions of Iran during 2007-2008 cropping seasons. Fifty one isolates were collected from different parts of Iran. Purification, inoculation and multiplication of isolates were conducted at the seedling stage of susceptible Bolani, under green house conditions. After inoculation on the seedlings of brown rust of wheat near isogenic lines, a total of fifty one pathotype were identified based on Avirulenve/Virulence formula. In two years some collected isolates showed similar virulence phenotypes included: TKTT, PKTT, PJTT, PHRR, PKRT, PHRT, PGTT and TCTT and the others were unique. Marivan I and Avaz II isolates showed the most virulence and Gorgan II was the least virulence in 2007. In 2008, Borojerd I and Ardebil I showed the more aggressive and Ahvaz II was the least. Among all the collected isolates during two years no virulence were detected on plants with Lr9, Lr19, Lr25 and Lr28 resistance genes. Eight and six clusters were identified based on drawn denderogram at 0.76 cutting line in 2007 and 2008, respectively.

In this present study the antagonistic effect of culture filtrate metabolites of twelve Trichoderma species including: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum, T. orientalis., T. brevicompactum and T. spirale was studied on Phytophthora sojae zoospore production and activity rate of β-1, 3-glucanase and β-1, 4-glucanase enzymes of all species in relation to degradation of Phytophthora cell wall used in laboratory condition. In final tests, the biocontrol effect of Trichoderma species on Phytophthora root rot of soybean in greenhouse, was also studied. To analyze the effects of culture filtrates on the zoospore production at concentrations of 0. 5, 1, 5, and 10 percent the culture were used in sterile deionized distilld water. To compare the effect of two types of carbon source on production of hydrolytic enzymes by Trichoderma, activity rate of β-1, 3-glucanase and β-1, 4-glucanase enzymes of all species were evaluated in presence of glycerol and P. sojae - hyphe as the carbon source in Wiendling’s medium. The results showed that culture filtrates of all species inhibited of zoospore production. Different concentration had different inhibition effects. The most inhibition effects was obtained from T. virens and T. brevicampactom culture filtrates. The results of enzyme assays showed that using Phytophthora - hyphe in medium increased the β-1, 3-glucanase and β-1, 4-glucanase enzyme activities compared with glycerol in all Trichoderma species. The highest activity rates for both enzyme obtained from T. brevicompactum and T. virens which for β-1, 3-glucanase in glycerol treatment were 2. 31 and 1. 73 U/mg protein and in P. sojae hyphe treatment were 4. 47 and 3. 91 U/mg protein. The activity rate of β-1, 4-glucanase was 0. 66 and 0. 86 U/mg protein in glycerol treatment and 1. 72 and 1. 72 U/mg protein in P. sojae hyphe treatment. To study the biocontrol effect of Trichoderma species on the soybean root rot severity, a completely randomized design was used with 15 treatments including each Trichoderma species cultured on wheat bran plus disease agent in sterile soil, at the trifoliolate stage (v1) of soybean (Wiliams cultivar), health control and disease control in the same kind soil, with three replicates at greenhouse condition. T. orientalis، T. brevicompactum and T. spirale showed the most effects on the disease severity reduction, damping of reduction and the growing factor promotion.

A wide range of pectolytic bacteria inflict damages in ornamental plants. Accurate identification and determination phenotypic and genotypic variation in populations of these bacteria are necessary to implement effective control methods. Thirty-eight isolates of leaf and fleshy parts of ornamental plants from different greenhouses in Guilan, Golestan, East of Mazandaran and Mashhad were collected. The phenotypic features, electrophoretic pattern of cell proteins and DNA fingerprints of the isolates were studied. According to the differential phenotypic characteristics, the isolates were assigned to Pectobacterium, Dickeya and Intermediate species. In numerical analysis of biochemical and physiological features, the isolates were divided to 12 groups. The cell protein pattern of the isolates was diverse and was only useful for preliminary grouping of the isolates. Genomic DNA fragments of representative isolates and eleven references strains were amplified with ERIC, BOX and REP primers. According to the combined dendrogram of the rep-PCR electrophorograms, the isolates were clustered to several groups at a low level of similarity to the reference strains. It seems that rep-PCR is not a useful approach for differentiation of pectolytic erwinias at species or subspecies levels.

During spring 2002, wild rocket (Sisymbrium irio) plants showing leaf spot symptoms were observed in Polor Tehran province. The spots were 0.5-1.5 mm in diameter, circular to irregular brown to black, and necrotic, surrounded by faint to conspicuous chlorotic halos. A levan positive pseudomonad was isolated from the symptomatic leaves on sucrose nutrient agar (NAS). The bacterium produced fluorescent pigment on medium B of Kings B medium. From the results of biochemical and physiological characteristics, the causal bacterium was identified as a pathovar of Pseudomonas syringae. Pathogenicity of selected strains was confirmed by inoculation of wild rocket with bacterial suspension of several strains isolated from wild. In electrophoretic profile of cell proteins the strains were, partially similar to the pathotype strains of Pseudomonas syringae pv. maculicola and P. s. pv. tomato. PCR amplification with the cfl primers yielded the expected 690 bp fragment from all strains. In ERIC-BOX and REP- PCR, the fingerprints of the strains from wild rocket showed 20% similarity to those of the standard strains of P. s. pv. maculicola and P. s. pv. tomato. Nevertheless, based on their overall characteristics the strains isolated from leaf spot on S. irio may be regarded as atypical strains of P. s. pv. maculicola, pending result of further genotypic analysis.

Grapevines in several locations in Fars province were sampled and tested for the presence of viroids. Total nucleic acid was extracted from leaves and subjected to RT-PCR using specific primers for reported grapevine viroids. PCR products were cloned and sequenced. Results showed that sampled vines were infected with Grapevine yellow speckle viroid 1 (GYSVd1), Grapevine yellow speckle viroid 2 (GYSVd2), Hop stunt viroid (HSVd) and Australian grapevine viroid (AGVd). GYSVd1 was the most abundant viroid while AGVd was the least frequent. No sample was found infected with citrus exocortis viroid. Iranian isolates of GYSVd1 were similar to the symptom inducing isolates of GYSVd1 and were associated with yellow speckle symptoms in the infected vines. However, severity of symptoms increased in the mixed infection with Grapevine fanleaf virus. Iranian isolates of GYSVd2 and HSVd were different from other isolates of these viroids in pathoginicity and left terminal domains. AGVd is reported for the first time from Iran. It is different from the known isolates of the viroid in biological and molecular properties.

Effect of different levels of nitrogen, phosphorus, iron and zinc on the activity of root-knot nematode (Meloidogyne javanica) on cucumber, cultivar Super Amelia, and the vegetative growth indices of the host plant, was investigated under greenhouse conditions. Combination of levels 0, 50, 100, 200 and 400 mg/kg of soil of nitrogen and 0, 25, 50 and 100 mg/kg of soil of phosphorous, from urea and triple superphosphate sources, respectively, also combination of 0, 2.5 and 5 mg/kg of soil of zinc and iron, from zinc sulfate and Fe-chelat sources, respectively were tested in 1.5 kg pots filled with field soil, in two independent experiments The experiments were done in randomized complete block designs, each with five replicates. Each pot was inoculated with five eggs or juvenile two/gram of soil, at six-leaf stage of plant. The results showed that application of 100 mg of nitrogen and 100 mg of phosphorus per kg of soil, also five mg of zinc and 2.5 mg iron per kg of soil caused a significant increase in the plant shoot length, shoot fresh and dry weight. The number of eggs, egg masses and galls were also decreased.

In this research, the effect of a defense elicitor, Bion ® (Acibenzolar-S-methyl) on fire blight disease in apple cv. Golden delicious was evaluated. Erwinia amylovora strains were isolated from blighted samples collected from Karaj, Qazvine, Mashhad and Neishabur. After identification using biochemical/ physiological tests and PCR, a mixture of four strains was used as inoculum. Golden delicious plantlets were propagated in MS medium supplemented with BAP and NAA and sprayed with Bion, distilled water and streptomycin and inoculated using two different methods. Based on the result, inoculation using shoot cutting method resulted in a higher blight severity than leaf cutting method. Having no bactericidal activity, Bion treatment reduced fire blight severity at a level comparable to streptomycin. Accordingly, it is conculded that Bion elicitor can control fire blight in the most prominent apple cultivar of the country, cv. Golden delicious.

In the course of studies on population dynamics and spatial distribution of the tea cottony scale, Pulvinaria floccifera (Westwood) (Hemi.: Coccidae) in the tea gardens of Tonekabon (west of Mazandaran), in 2008-2009, infected cottony scales were found with hyphae and orange-flask shaped perithecia. They were grown separately on PCA and kept under isolated conditions and in incubator at 24-25ºC. After 10 days they were sent to Plant Protection Research Institute of Iran for further investigation. The collected pathogens were as follows: Lecanicillium muscarium (Petch Zare & Gams), Lecanicillium lecanii (Zimmerm. Zare & Gams). Lecanicillium lecanii: Colonies reach 15-25 mm in diam. in 10 days, being rather compact, yellowish white, with deep yellow reverse. Phialides which are relatively short, 11-20(-30)× 1.3-1.8 µm, aculeate and strongly tapering, are produced singly or in whorls of up to 6 directly on prostrate hyphae, or on short, more or less erect conidiophores. They are sometimes also produced secondarily on the previous phialides. Conidia are formed in heads at the apex of the phialides, typically short-ellipsoidal, 2.5 -3.5(-4.2) ×1-1.5µm, homogeneous in size and shape. Octahedral crystals are also present. Lecanicillium muscarium: Colonies reaching (14-) 25-30 mm in diam in 10 days, being rather compact, white, with cream-coloured to pale yellow (rarely yellow) or uncolored reverse. Phialides are produced directly on prostrate hyphae or on secondary branches. Secondary branches are less frequent than in L. lecanii. Phialides are generally longer than those of L. lecanii and less tapring, measuring (15-) 20-35× 1.0-1.7 µm. Conidia, are produced in globose heads, ellipsoidal to subcylindrisal, more irregular in size and shape, longer and narrower than in L. lecanii, measuring (2-)2.5-5.5(-6)×1-1.5(-1.8)µm. Octahedral crystals commonly present (IRAN 1650C). Torrubiella. cf. conferagosa: Mycelium is thin, white to cream color, covering scale insect and extending slightly beyond on the substratum, slightly tufed, pulverulent; perithecia are irregularly scattered to crowded over the scale, superficially or slightly embedded at the base in the mycelium. Asci are ovoid and cylindrical, and ascospores are filiform (IRAN 14441F).

Strains of Spiroplasma citri have been classified into five groups based on electrophoretic mobility (EM) of spiralin protein and RFLP analysis of spiralin gene (Foissac et al. 1996). In the present study, two isolates of S. citri qualifying for formation of a sixth group are reported. Infection of sesame plants by S. citri has been reported previously (Salehi and Izadpanah 2002). Samples of vector (Circulifer haematoceps) leafhoppers (Salehi et al.1993) were collected in various regions in Fars province of Iran. The insects were caged on periwinkle (Catharanthus roseus) plants. Presence of S. citri in collected plant samples and vector-inoculated plants was verified by ELISA using a locally produced polyclonal antiserum against a citrus isolate of S. citri. ELISA-positive samples were used to isolate S. citri in culture medium LD10 (Lee and Davis 1984). S. citri grown in culture was used to amplify spiralin gene by PCR using specific primer pair F1/R1 (5´ -TAATTTTAATAACATTTGCTT-3´/5-´ GTTCTAAATAAGAAAAAGTTT-3´). The amplified products were cloned in E. coli and sequenced. RFLP analysis showed that most isolates fell into group I of S. citri strains but two isolates originally obtained from C. haematoceps in a sesame field and propagated in periwinkle (Catharanthus roseus) did not match any of the five groups. The spiralin gene in both isolates (Acc. No. FJ755921 and FJ755922.) had only 93 percent homology with that of GII3 strain and could code for 244 amino acids (aa) instead of 241 aa in strains of groups 1-4 and 242 aa in a group 5 (Palmyre) strain. RFLP analysis showed that the leafhopper isolates had one additional MboI restriction site at nucleotide 456 plus changes in the position of certain other restriction sites. These results were confirmed by electrophoresis in polyacrylamide gel in which the spiralin protein from both insect isolates showed a different EM and banded at 28.8 kDa compared to 32 kDa for palmyre isolate and 24-28.5 reported for groups 1-4. By the current criteria (Foissac et al. 1996) the two new isolates qualify for forming a new group (group 6) of S. citri strains from Iran.