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.

Pistachio gummosis disease caused by Phytophthora species and its control management with soil solarisation method in Iran.

Pistachio (Pistacia vera L.) is the most important commercial product in Iran and root rot or crown rot (Gummosis) is the most serious diseases of this crop. During 2005-2007 Infected trees of Pistachio orchards were visited and plant samples plus soils around the infected trees collected from Kerman province in Iran. Samples were transferred to laboratory and cultured on common medium and using citrus leaves pieces as baits on water-saturated soils. Different Phytophthora species were isolated and studied to be identified. Three Phytophthora species including P. megasperma, P. drechsleri and P. citrophthora were the principal cause of pistachio gummosis and root rot in Iran. However, Phytophthora pistaciae as new species was introduced as aggressive species to different Pistachio cultivars. Since chemical control was not property managed the disease, soil disinfestations by soil solarisation method was carried in Kenrman as the nearly warmer climate in studied areas to manage the pathogen. Application of this method reduced population density of the pathogen from 1300 to 200 CFU -g/soil after 6 weeks. This method was effective, non negative side and economic which can be used in all agricultural areas.

Major Fusarium diseases on crops and their control management with soil solarisation in northwest Iran.

Fusarium species are the most frequently soil-borne fungal pathogens on crops that make high economical damages in Iran. Studies showed that Fusarium species cause significant yield losses in main crops especially potato, pea, bean, wheat, corn and rice in northwest Iran. The diseases resulted in yield losses to the extent of 30% to 70% in the fields and made economical problems for growers. Infected plants were collected and cultured common medium (PDA) and selective media (PPA, CLA) for Fusarium species after surface sterilization. The dominant species were F. solani, F. oxysporum, F. graminearum, F. moniliforme, F. sambucinum, F. culmorum, and F. equiseti in area studied. Soil solarisation method was carried at the summer season in three soil infested locations to assess the control management of the pathogens. Application of this method reduced population density of the pathogen from 1900 CFU -g/soil to 500 after 4 week. This proper method was simple, effective, non negative side and economic which can be used in nearly different farming areas at warm season.

Biological control of Orobanche aegyptiaca by Fusarium oxysporum F. sp. Orobanchein northwest Iran.

Broomrape (Orobanche aegyptiaca) is one of the most serious weed pathogen on many plants all over the world, especially in northwest Iran. It causes damage on some plants particularly on tomato, cucumber and other dicotyledonous crops in zanjan province. Broomrape as weed, caused reductions in crop yield, adversely affected crop quality, and resulted in loss of cultivated land due to reduced crop alternatives. Since there were no any chemical methods and other proper technique to control this plant parasite we tried to find a good mechanism for its management in the fields. Our study showed there was a specific species, Fusarium oxysporum f. sp. orobonche that had infected Broomrapes naturally in the field. In point of fact the species was isolated from naturally infected Broomrape in studied Locations. Our surveys showed F. oxysporum f. sp. orobanche caused disease on orobanche spp. in different agricultural fields. Although there were other fungal species which can nearly manage the orobanche but it can be fungal pathogen to other plants. However the best fungal isolate can be F. oxysporum f. sp. orobanche since it may not be pathogen for other plants.

Wilting of date palm branches by Fusarium oxysporum in south of Iran and its control managements with soil solarization method.

Wilting of some branches in nurseries and orchards of date palm were studied in south of Iran including Ahvaz and Abadan cities in 2005-2006 years. Different infected plants were visited and samples showing symptoms including wilting or death of branches collected from various areas and transferred to laboratory. Samples were cultured in common media (PDA) and different fungi were studied and identified. The most frequently isolated pathogen was Fusarium oxysporum which caused wilting of some branches of date palm seedling or trees in studied areas. Results showed that the disease caused main losses where date palm cuttings were cultured in infected soils, previously cropped to susceptible plants. Since chemical control was not managed the disease, soil disinfestations by soil solarization method was carried in Ahvaz as the warmer climate in studied areas to control the pathogen. Application of this method reduced population density of the pathogen from 1800 CFU -g/soil to 600 after 5 week. This method was simple, effective, non negative side and economic which can be used in nearly warm areas.

Mycotoxin producing Fusarium species associated with plant disease on potato, wheat, corn and animal diseases in northwest Iran.

There were some plant diseases on potato, wheat, corn, bean and animal diseases such as feed refusal, weight loss, death of cattle and sheep as well as chicken mortality in northwest Iran. Infected plants were collected and cultured in PDA as common medium and Peptone PCNB Agar (PPA) as selective medium for Fusarium species after surface sterilization with sodium hypochlorite. Several Fusarium species were isolated from samples counting potato tubers, wheat, corn, plant residues and animal feeds in the fields and storages. Actually, Fusarium species were the major pathogens causing significant diseases on potato, bean, wheat, corn, rice and alfalfa as the key human food and animal feed in that areas. Study showed most plant and animal diseases especially chickens mortality were attributed to feeding infected plant straw and contaminated feeds in considered areas. Mycotoxin producing species including F. solani, F. oxysporum, F. graminearum, F. moniliformei, F. sambutinum, F. culmorum and F. equiseti were dominant recognized isolates. The common Fusarium mycotoxins such as zearalenone, moniliformin and fusaric acid have been also discovered from these species. The results put emphasis that Fusarium contamination of feeds or foods can be capable of the harmful consequences on animal and human health.

Verticillium wilt of olive branches in Iran and its control managements with soil solarization method.

Wilting of mostly one branch in nurseries and newly established orchards of olive was studied in eastern north of Iran including Zanjan, Golestan and Khorassan provinces during 2002-2004. Different infected plants were visited and samples showing symptoms including wilting or death of branches collected from various areas and transferred to laboratory. Samples were cultured in common media (PDA) and different fungi were identified. The most frequently isolated pathogen was Verticillium dahliae which caused wilting of mostly one branch of olive seedling or trees in studied areas. Results showed that the disease caused main losses where olive cuttings were cultured in infected soils, previously cropped to susceptible plants. In fact, the population density of V. dahliae was high in the garden soil, previously produced susceptible crop cultivar to the fungus especially cotton or potatoes. Soil disinfestations by soil solarization method was carried in Taroam as the warmer climate in studied areas to control the pathogen. Application of this method reduced population density of the pathogen from 1700 CFU g/soil to 1000 after 4 weeks. This method was simple, effective, non negative side and economic which can be used in nearly warm areas.

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.

Severity of walnut anthracnose and its relatively resistant in Iran.

Walnut black spot or Anthracnose has been a destructive disease of Juglans in Iran mainly northwest of the country. Current situation of the disease was studied in various regions including Qazvin, Zanjan, Hamedan and East Azarbyjan provinces during 1999 to 2002. Infected samples such as leaves, fruits and foliage were collected and cultured in PDA, CMA and NA media after surface sterilization with sodium hypochlorite. Morphological characteristics and asexual reproduction of isolated pathogen showed that the fungal causal agent was Marssonina jglandis (Lib) Magn. which perfect stage was Gnomonia leptostyla (Fr). The disease was severing under studied areas, especially when it was rainy and humidity. Virulence of the disease caused major defoliation in some walnut trees infected to anthracnose disease. So, investigation showed that the collecting or burning infected leaves and fruits under trees could reduce severity of disease. Study also resulted there was a resistant variety around the Qazvin province traditionally called "Alamoty" which had not any anthracnose infection in natural condition. The result of experimental work with inoculation of different walnut clones by spore suspension (10(5)) in glasshouse has also indicated that this original clone was more resistant than others to anthracnose disease. Alamoty clone had favorite yield production and some trees presented more than 250 years old in Iran.

Pathogenicity of two species of Fusarium on some cultivars of bean in greenhouse.

Twenty isolates of Fusarium oxysporum and F. solani were isolated from the infected roots of bean in different farms of east Azarbaijan and Tehran Provinces and their pathogenicity determined. Most isolates of the fungi were identified as F. oxysporun. They caused root rot, yellowing and wilting of bean in the field. In this test, the roots of 6 cultivars of bean seedlings soaked in suspension of the 7 isolates of the fungi (a1, Gogan, a2, Bilverdi, a3, Savojbolagh-Hashtgerd, a4, field of Agr. Coll. a5, Khomein, a6, Ramjin of F. oxysporum and a7 of F. solani of Varamin, Iran) for 5 minute (106 spores/ml.) then transplanted into the sterilized soil in 4 pots (as replication). For control (a8) the roots soaked in distilled water. The results showed that percentage average of necrotic roots and crowns of isolates al, a2, a3, a5, a6, a7 was %20.31 in group a, a4 was %43.52 in group b and a8 was %2.77 in group c after 3 weeks. The isolate a4 (from the field of Agricultural College, Karaj) was more infectious than the other because it caused wilting, yellowing the leaves and decreased the growth very soon, followed by a5 with %25.32 rate was more pathogenic. Bean cultivar Goli-Red was more tolerant with %10.02 than the others of 16.29 (Naz Red) to 25.15 percent of necrotic the roots & stems.

Susceptibility of six cultivars of bean to different isolates of two species of Fusariumin greenhouse.

In greenhouse, susceptibility of 6 cultivars of bean (b1, green bean cv. contender, b2, Pinto b. cv. Cos-16, b3, green b. Sun Ray, b4, white b. cv. Daneshkadeh, b5, red b. cv. Naz, b6, red b. cv. Goli) evaluated to 7 isolates of 2 species of Fusarium (6 isolates of F. oxysporum and 1 isolate of F. solani from Varamin, Iran) in 4 pots (as replications) in factoriel experiment. The seedlings of each bean cultivars soaked in a suspension of the fungi (106 spores/ml.) for 5 minutes and transplanted into the pots contained sterilized soil, for control, the roots soaked in distilled water. After 35 days of inoculating and transplanting in greenhouse conditions (with 18-26 degrees C and 12 h light & 12 h dark) the percentage of necrotic roots and stems of the seedlings affected to the isolates of the fungi, also height and weight of them in different treatments datermined and calculated. The results showed that, the average of necrotic percent of the cultivars of bean seedlings grouped in 3 category (b1, b2, b3, b4 with 25.15% in group a and b5, red bean cv. Naz with 16.29% in group b and b6 red bean cv. Goli with 10.02% in group c) based on cluster analysis. But by Dunken method the infection set on 5 groups as respectively b4, 28,72% (a), b2, 25.83% (ab), b1 & b3, 23,18% & 22.84% (b), b5, 16.29% (c) and b6, 10.02% (d). So, red bean cv. Goli was more resistant and white bean cv. Daneshkadeh was more susceptible than of another cultivars, but averse the effect of the fungi on growth factors were different.

The causal agents of damping-off disease of buglosse from Iran.

Iran is considered a major genetic for medicinal plant in the world. Because of this significant diversity and historical background in identification and utilization to remedy human and animal diseases, export of medicinal plant can help to strengthen local as well as natural economy. Buglosse (Fig. 1) is one of the most important and common medicinal plants in Iran and exist as Echium amoneum and Borago officinalis. This work was conducted in order to identify the causal agent(s) of damping off disease in buglosse. Plant disease samples were taken from Esfahan and Tehran provinces. Symptoms on original plant including root, crown rot, dark tissue, pith and hallow root were collected in order to isolate disease agent(s). Symptomatic root and crown tissues after surface sterilization with 96% ethanol were transferred on to PDA and WA media and also on moist filter paper in petri dishes. Two fungal colonies grew from tissue segments and spore culture was subsequently purified. The fungal isolate identified as Rhizoctonia solani based on the following test. Hyphal tip was removed from colony margin placed on PDA and PSA media and incubated in dark. Colony diameter of one hundred hyphae measured and nucleus was stained according to Bandoni (1979), Kronland and Stanghellini (1988). It was observed that in each cell of hyphae there are more than two nuclei. Single spore culture were obtained from macroconidia of Fusarium isolate. After 24 hr of incubation, growing single spore were transferred to KCL medium to detect spore chains. Fungal isolates transferred to PSA and PDA media for sporulation. After 7 days colonies appeared as white cream to pinkish on top and cream to dark pink at the bottom of petri dish with abundant micro and macro conidia. Colonies were snow white, felting shape, with ample causal hyphae on PSA medium. On KCL medium, fungal growth was superficial and colonies were colorless with long macroconidia and individual sausage-shape macroconidia being thinner one side and having maximum four septa. Microconidia were long double compartment round on both side, straight to slightly curved. Base on morphology and dimension of conidia and production of chlamidospore the funguses identify as Fusarium solani.

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.

Study on the overwintering of cleistothecia and conidia of Erysiphe betae causal agent of sugar beet powdery mildew in Iran.

Sugar beet leaves covered by sexual (cleistothecia) and asexual forms (mycelia and conidia) of Erysiphe betae were gathered at harvest time and maintained under natural outdoor conditions. In order to determine the function of cleistothecia and also conidia in the overwintering of E. betae some experiments were performed. The results showed that ascospores were unable to be released in petri dishes but their release under natural conditions occurred after 4 months. Under In vitro conditions ascospores did not germinate but on the leaves germination was rarely possible, however these ascospores were degraded after 7 days and didn't have pathogenicity. Conidia could induce pathogenicity after 3 but not 4 months. The period after inoculation until appearance of disease symptoms increased with aging of conidia. The results for conidial germination showed that fresh conidia had 80 percent germination on glass slides but it decreased sharply after 2 weeks and reached to 0 percent after 4 weeks. Although their germination on the leaves was not more than 46 percent of fresh conidia but they had good germination after 2 and 4 weeks. The results for the experiment to observe the first appearance of the disease in the field suggested that the first conidia were trapped by spore-trap in early June and the first symptoms appeared 20 days later. The conclusive results showed that ascospores had no function in the survival of the fungus and air-borne conidia are the main source of primary infections.

Chitinase gene transformation through Agrobacteriumand its explanation in soybean in order to induce resistance to root rot caused by Rhizoctonia solani.

Chitinase gene (chi) of bean which has been cloned in recombinant binary plasmid vector, pBI121 with 35s promoter of Cauliflower mosaic virus (CaMV), were used for transformation of soybean using strain LBA4404 of Agrobacterium. The plasmid contained nptII gene that is a resistant gene to kanomycin as selector marker and Gus gene as reporter. Cotyledon explants of Williams and Clark cultivars were inoculated by Agrobacterium suspension with pBI121 and were cultured in regeneration medium. After complete regeneration of explants to seedling in B5 medium amended with kanomycin, polymerase chain reaction analysis were conducted to ensure conjugation of nptII, Gus, CHN genes in transformants seedling of soybean. Results showed that some lines of soybean contained Gus and CHN genes. More ever, chitinase activity in leaf extract of transformed soybean lines was significantly more than untransformed soybean, exception one sample. Bioassay of chitinase activity of transgenic lines on in vitro condition prevented mycelial growth of Rhizoctonia solani in comparison with untransformed control leaf extract.

Occurrence and physico-serological properties of potato virus S (PVS) in Iran.

1818 collected samples of potato plant showing virus infection symptoms from 85 fields were tested for PVS infection using DAS-ELISA. Average of infection to this virus varied from 0 to 100%. Least infection was belonging to fields with new introduced varieties. On the other hand native and old introduced cultivars showed heavy infection. In field condition, PVS infected plants didn't show very obvious symptoms, so some infected plants may be missed in field sample collecting. The physical properties of 3 isolates, Avaj, Stanboly and Agria No 15 were determined. TIP 55-60 degrees C, DEP 10(-3) and Liv measured 3-4 days. Ouchterlony agar double diffusion test using SDS was useful for virus detection and precipitation lines didn't show any spur between isolates, although isolates differs slightly in symptomatology. SDS-Page and Western blotting methods used successfully for virus detection and determining and measuring viral protein components.

Determination of the races of potato virus X and its host rang in Karaj, Damavand and Ardabil, Iran.

Potato virus x (PVX) is found commonly in potato-growing areas, worldwide. It is an economically important virus which causes losses in tuber yield of approximate 5 to 15 percent. In a 2 year survey, potato leaf and tuber samples were collected from various fields in Damavand and Karaj. The initial isolations from potato were made by mechanical inoculation first to Gomphrena globosa L. and later to Dartura stramonium L. It was not transmitted by 2 species of Cuscuta but transmitted mechamically. The isolates were inoculated to Nicotiana glutinosa L. in which they were maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, after 10 minutes at 70 degrees C and after 10 weeks at room temperature. The virus had filamentous and slightly flexuous particles with a normal length of about 490-500 nm and 12 nm width. According to the symptoms, TIP results and serological comparisons, the compared isolates showed no difference and they belong to XN group. In order to estimate disease incidence, 773 tubers from Damavand area were tested and compared with that in Ardabil area. Disease incidence in Damavand ranged from 1.1-20.9 percent and was lower than disease incidence in Ardabil. In 8 genera of collected weeds from fields of potato and tomato samples by using test plants and serological methods, they didn't show existence of the potato virus x.

Study on potato virus M (PVM) occurrence in potato fields in Iran.

57 native potato tuber samples collected from different potato growing region of Iran, planted on single rows in Karaj College experimental station. Plant samples of each single row plus 9.25 Fresh foliage samples collected from fields under new introduced cultivars were tested for potato virus (PVM) infection during growing season. Also 78 weeds and field crops belonging to Solonacae and Leguminosae from or neighboring to potato field were tested. Results indicated that PVM was not found on any plant other than potatoes. PVM was detected on 16 samples of 57 old vars, Virus was not seen in any samples collected from fields under new varieties. Results show that PVM is limiting in this crop. PVM detecting is difficult using assay hosts. Best test plants were French bean var Red kidney, Showing pinpoint necrotic LL, also Datura metel and Nicotiana debneyi are useful for virus detection showing chlorotic local lesion. Also microprecipition and gel diffusion test can be used for virus detection but Elisa was the best method. PVM infected plant showed 11-19.5 percent yield decrease in 3 cultivars tested.

Study on infection symptoms of root-knot nematode, Meloidogyneja vanica, on the stem of the tomato seedlings in greenhouse.

On the first of seeds germination, Meloidogyne javanica induced knots on the stem eventually by to penetrate into the coleoptiles. In this experiment, used soil was loamy-sandy type, pH=7.2, including 1.5% composts. Tomato seedlings of cultivar Rutgers were used as host. This experiment was conducted in Completely Randomized Design with 3 treatments and 10 replicates as fallow: (1) control, (2) 10 J2s/cm3 of soil, (3) 20 J2s/cm3 of soil. In each pot (1000 cm3), 50 seeds were planted. Inoculation was done, a day after to plant seeds, in 2 cm depth. Pre-emergence and post-emergence damping-off, number of stem-knots, height of knots on the stem in 3 treatments was more than 2 treatments. In some of the pots of 2 treatments were not observed stem-knots. As long as four weeks after planting, there was not significant difference in fresh weight and height of the seedlings in treatments. Numbers of adult females in 3 treatments were more than 2. In the stem-knots, collenchymas tissue, endodermis and stem cambium were found deformed J2s and J3s, and adult females.