مجله دانش گیاه پزشکی ایران
سال چهل و دوم شماره 2 (پاییز و زمستان 1390)

Aflatoxin is one of the most hazardous mycotoxins injurious to both humans and animals. Aflatoxin contamination of crops is a serious economic loss inflicted upon agriculture, food and animal industry throughout the entire world. In nature, there are microorganisms able to alleviate aflatoxin contamination in crop products. In this work, the effects of four strains of Bacillus subtilis in reducing aflatoxin, induced by Aspregillus flavus, were investigated under in vitro conditions. The results indicated that strain BsP1 could completely inhibit the mycelial growth of A. flavus in PDB liquid medium. Strains BsP4, BsP5 and BsP38 caused a mass reduction of 58.88, 27.41 and 29.03% of fungal biomass, followed by 86.85, 21.80 and 26.16% reduction of aflatoxin B1 (AFB1), respectively. There also was a negative significant correlation observed between the decrease in fungal biomass and AFB1 production. Bioremediation studies revealed that the bacteria didn’t have the ability to remediate AFB1 after 3 days of incubation. However, the strain BsP1 could completely remediate AFB1 even after 5 days of incubation. Cell walls of these bacterial strains showed no sign of an ability for binding and removing of AFB1. The supernatant fluid of strain BsP1 was considerably able to degrade AFB1. The results finally suggest that the promising isolate of B. subtilis is of the potential to reduce aflatoxin in susceptible crops, possibly through antibiosis and degradation mechanisms.

One of the most importanat traits of fluorescent pseudomonads, a phenomenon also related to their biocontrol, is the ability to develop phase variation during their rhizosphere colonization. For an investigation of this phenomenon, at first 24 bacterial strains of Pseudomonas fluorescens that were able to cause an inhibiting zone within the range of 3-14.67 mm. against Gaeumannomyces graminis var. tritici (Ggt) were initially selected for greenhouse experiment. Following the greenhouse experiment, 12 bacterial strains, able to control the take-all disease within a range of %47.5-%77.5 were chosen for studying the phase variation phenomenon in laboratory conditions. The 12 bacterial strains were cultured on King B and SA media on seven of which, viz, Um141, Um11, Um138, Um70, Um115, UmCHN5 and Pseudomonas fluorescens F113 colonies of various morphologies were observed. Results of phase variation phenomenon during rhizosphere colonization in these bacterial strains (both in sterilized conditions and in the presence of Ggt), showed that phase variation had occurred in these strains during the process of rhizosphere colonization and these strains had been able to adopt themselves to root in various growing conditions and as well to penetrate to the end of the root. A comparison of bacterial population in different parts of the root indicated that the most population prevailed at the bottom of the root area. The results of the phase variation phenomenon in the presence of Ggt proved the fact that this phenomenon can lead to a reduction of the rate of take-all disease.

To provide reliable information concerning reproductive potential of Hippodamia variegata Goeze and its prey (Aphis fabae), their related life tables were studied under controlled laboratory conditions (at 23ºC). The unprocessed data of life tables were analyzed taking the Euler-Lotka as the basis model. Standard error of population growth parameters was calculated using the Jackknife re-sampling method. The Mann-Whitney U test was utilized to determine differences in population parameters of H. variegata and of A. fabae. The intrinsic rate of increase (rm) of ladybird was 0.197±0.002d-1. Other table parameters, including net reproductive rate (R0), mean generation time (T), the finite rate of increase (λ) and the Doubling Time (DT) were estimated as 365.43±22.09 offsprings, 29.96±0.37 d, 1.218±0.002 d-1 and 3.52±0.033 d, respectively. These parameters were estimated for A. fabae as: rm= 0.309.±0.01 d-1, R0= 41.98±4.6 offspring, T = 12.1±0.21 d, λ= 1.36±0.015 d-1 and DT=2.24±0.078 d. A comparison of the two life table parameters showed that there existed significant differences between life table parameters of H. variegata and those of A. fabae. In all cases, the aphid exhibited a more prominent reproductive trait.

Aphids are among important agricultural pests. The pest's evolutionary tendency towards a loss of taxonomically useful characteristics, as well as morphological plasticity due to host type and as a result of environmental factors, make a morphological identification of aphids difficult, whereas exact identification is indispensable for managment of these pests. Thus, modern identification methods could help in managing these pests. In this study conducted during 2008-2010 on trees in pome fruit orchards in Mashhad region of Iran, classic method accompanied with DNA barcoding was employed for an identification of aphid pests. Aphis pomi, Dysaphis afiinis, D. plantaginea, Nearctaphis bakeri and Allocotaphis quaestionis were collected from apple, quince and pear trees, A. quaestionis being reported for the first time from Iran. DNA barcoding was provided for cox1 gene in these species and phylogenetic relationship investigated among the collected species as well as other species within the family. DNA barcoding confirmed the morphological identification. Results of molecular data were compatible with morphological characteristics and classic grouping, separating the studied species as based on genus and tribe. This is the first data gathered about aphid characterization in Iran while applying DNA barcoding. The results of this study indicate that cox1 sequence could be useful in aphid's identification and recognition of their internal relationships.

Thirty five monoconidial isolates of the fungal culture collection Pyricularia grisea were examined, to identify the vegetative compatibility groups and characterize the genetic diversity of isolates as through rep-PCR genomic fingerprinting. The isolates were collected from ricefields during 1997-1999. Genetic diversity of P. grisea isolates was studied as based on DNA fingerprinting through rep-PCR using two primers previously designed based on the nucleotidal sequence in ERIC and BOX regions. They generated variable length fragments ranging from 400 to 2500 bp. Phenetic analysis let to differentiation of four distinct clonal lineages designated as A to D. Clonal lineage A with 74.28% frequency, constituted the largest fingerprinting group. For VCG analyses nit mutants were obtained from fast growing sectors on Minimal Medium (MM) containing 5% potassium chlorate. Complementation between nit mutants of isolates was tested on MM. Four vegetative compatibility groups were determined including VCG1, VCG2, VCG3 and VCG4. Group VCG3 comprised of 14 isolates was the dominant VC group among others. This study revealed that isolates obtained from rice were designated in the four VC groups forming heterokaryon with each other; however these isolates were separated with more than 80% similarity from each other using rep-PCR marker with most of isolates being VCG3 were determined in A clonal lineage. Both VCGs and rep-PCR analyses determined low genetic diversity in P. grisea population from rice.

Eucalyptus camaldulensis Dehnh and Callistemon viminalis (Gaertn) essential oils were tested against adults (1-3 day old) of Tribolium confusum Jacquelin du Val. in 280 ml glass vials either with or devoid of wheat grains. The oils were also examined for fumigant toxicity against, 5 and 20-day-old larvae. The essential oils were obtained through hydrodistillation, applying a modified Clevenger type apparatus. Experiments were carried out at 27±1°C and 65±5% R.H. under dark conditions. Results of fumigant toxicity tests indicated that mortality increased with increase in concentration and time. Lethal concentration of E. camaldulnsis and C. viminali 50% of adults (LC50) were estimated as 20.88 and 20.06 µL/L air, respectively. Lethal times to kill 50% of adults (LT50) at concentrations of 35.71, 32.14, 28.57, and 25 µL/L air lasted 20.59, 15.94, 14.35 and 13,39 h for E. camaldulnsis oil whereas 17.57, 14.46, 11,69, and 10.72 h for C. viminalis, respectively. The most sensitive stage was 5-day-old larvae followed by adults of T. confusum, while 20-day-old larvae were highly tolerant to the tested essential oils. The lethal effect activity of the essential oils decreased significantly in jars filled with wheat kernels. LC50 values of E. camaldulnsis, and C. viminalis to kill T. confusum adults were 878.50, and 741.27 µL/L air, respectively; showing the low fumigant efficacy of the oil in the presence of grain. This may be due to a considerable sorption of essential oil by wheat grains and insufficient permeation of the oil vapor into seed inter-spaces.

To determine the specificity and host range of Wilsonomyces carpophilus isolates, some stone fruit seedlings (Prunus armeniaca, Prunus dulcis, Prunus reuteri, P. vulgaris, P. domestica, P. persica, P. avium and P. cerasus), pome fruits (Malus communis, Pyrus communis, Crataegus azarollus and Crataegus monogyna) as well as ornamental plants (Rosa persica, Rosa sp. and R. damascena) were selected for pathogenicity tests under greenhouse conditions. Five isolates from five different hosts (apricot, almond, plum, peach and sweet cherry) were taken for pathogenicity tests on ten leaf-seedlings under 20±2˚C and relative humidity of more than 75%, using atomized 105 conidial suspensions. The results showed that all isolates caused disease on Prunus seedlings. No specificity was observed between isolates and hosts. Pale brown to black lesions were observed on some of the pear leaves and twigs, and as well on apple and hawthorn leaves after 4 days past of inoculation. Lesions did not expand or abscise and there was no sporodochium observed on leaves even after 20 days past, so that, pear, apple and hawthorn were not determined as important hosts of the fungus. In addition, symptoms were not observed on the ornamental plants and on whitethorn, raising the expectation that the fungus is limited to stone fruits. Appearance of disease symptoms on almond, plum, peach and nectarine, and on apricot twigs in orchards, infection of plum and apricot twigs in greenhouse and isolation of fungus from these are the first reports from Iran. To evaluate the relative resistance of nine peach cultivars to tree fungus isolates, as based on the number of lesions per 10 cm2 of leaf surfaces, tests were carried out under greenhouse conditions (20±2˚C and relative humidity of more than 75%). The results indicated that there were significant differences among relative resistance of cultivars and among isolate's pathogenicity. Redtop, Springcrest and Early Elberta cultivars were resistant, whereas, Dixired cultivar was recorded as highly susceptible.

To identify the horticultural of plant parasitic nematodes in Northern Khorasan province, 170 soil and plant root samples were collected during different seasons of years 2009 and 2010. Nematodes were extracted through centrifugal-flotation method, specimens then being fixed according to modified Seinhorst method and transferred to glycerin before making measurements (De Grisse, 1969). Throughout the study 12 species from genus Filenchus namely: F. afghanicus (Khan & Khan, 1978) Siddiqi, 1986, Filenchus andrassyi (Szczygiel, 1969) Andrassy, 1979, F. baloghi (Andrássy, 1958) Siddiqi, 1986, F. cylindricaudus (Wu, 1969) Siddiqi, 1986, F. facultativus (Szczygiel, 1970) Raski & Geraert, 1987, F. misellus (Andrássy, 1958) Raski & Geraert, 1987, F. paravesiculosus Karegar & Geraert, 1998, F. polyhypnus (Steiner & Albin, 1946) Meyl, 1961, F. sandneri (Wasilewska, 1965) Raski & Geraert, 1987, F. vulgaris (Brzeski, 1963) Lownsbery & Lownsbery, 1985, F. elegantulus Raski & Geraert, 1986, F. longicaudatulus Zell, 1988 were identified from which the last two species are new /recirds for nematode fauna of Iran.

Fumigant toxicity of essential oils from Foeniculum vulgare (Apiaceae), Teucrium polium (Lamiaceae) and Satureja hortensis (Lamiaceae) were evaluated against the adults of cowpea seed beetle, Callosobruchus maculatus exposed for 24h. Essential oils were obtained through steam-distillation using a Clevenger-type apparatus. Bioassays were conducted at 28±2 °C and 60±5% R.H. in dark conditions. Six concentrations of each oil were utilized each in six replications. Gas chromatography coupled with mass spectrometry was applied to identify the main components of the essential oils. The principal constituents of F. vulgare oil consisted of E-anethole (60.61%) and Fenchone (12.14%) while those of T. polium oil were piperitenon oxide (21.72%), α-pinene (11.33%) and carvon (11.29%). S. hortensis oil mainly consisted of carvacrol (50.13%) and thymol (27.77%). All the essential oils exhibited high fumigant activity against C. maculatus adults. The results indicated that mortality of 1-day-old adults increased with oil concentration. Males were more susceptible to the oils than females. The essential oil extracted from F. vulgare proved to be most toxicant with LC50 values of 21.31 and 29.45 µL L-1 of air against male and female pests, respectively. The LC50 values of T. polium oil were recorded as 54.63 and 80.75 µL L-1 of air, while the corresponding values of S. hortensis oil were 74.36 and 156.66 µL L-1 of air against male and female pests, respectively. The results demonstrated that the essential oils of these plants, especially those of F. vulgare can be considered as potentially effective control agents against cowpea seed beetle.

Two separate greenhouse experiments were conducted in the greenhouse of the Iranian Plant Protection Research Institute, Tehran, to compare the efficacy of some Acetyl CoA Carboxylase (ACCase) and Acetolactase Synthase (ALS) inhibiting herbicides in control of wild oat biotypes. In each experiment, populations were independently treated by 10 herbicides each in 12 replications. To evaluate the effects of treatments, different characteristics including percent damage as based upon EWRC scores within 15 and 30 days after spraying, percent survived plants after spraying relative to that before being sprayed, as well as percent dry and wet weights of individual plants, relative to control, were scrutinized. Results indicated that the biotypes of wild oat were best controlled by clodinafop propargyl, and among ALS inhibitors, iodosulfuron plus mesosulfuron plus mefen pyre sulfosulfuron+metsulforon could control biotypes of Avena ludoviciana. very effectively vs. semi-satisfactorily, respectively. Iodosulfuron plus mesosulfuron and sulfosulfuron plus metsulfuron could dramatically reduce the wet weight of individual plants as compared with control so that the plants were not damage inflicting any more.

The antagonistic effects of Rhizobium spp. isolates against Rhizoctonia solani (AG-4), the causal agent of bean damping-off were compared with each other in either of the in vitro and in vivo conditions. Also their potential to produce such secondary antifungal metabolites as HCN, siderophore and protease were evaluated. The results indicated that, isolate RH3 reduced mycelial growth of R. solani more than the others did in vitro conditions. Isolates RH4, (RH6, RH3) and RH6 produced the highest siderophore, protease and HCN, respectively. Under greenhouse conditions, strilized soil was used to evaluate the effect of seed coatings with bacterial isolates on disease incidence, percentage of disease control and as well on growth factors. Isolates RH5 and RH4 indicated the highest level of plant dry weight in the presence of pathogen. Although none of the bacterial isolates could suppress the disease completely under greenhouse conditions, RH3 isolate could control the disease to an extent more than 80%. The obtained results indicated that the biocontrol potential of Rhizobium isolates not only led to an alleviation of the disease but also promoted plant growth. Then such bacterial isolates can be successfully applied in sustainable agricultural production systems.

The non-defoliant strain of Verticillium dahliae (SS-4) was isolated from olive groves showing disease symptoms in Toshan area of southern Gorgan city. Root-knot nematode, Meloidogyne javanica, was recovered from infested olive seedlings and following identification it was propagated on tomato seedlings cv. Rutgers. One-year-old seedlings of olive cultivars, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. The experiment was conducted in a completely randomized design of 32 treatments and five replications. The treatments included: control, nematode alone, fungus alone and fungus+nematode. Pots (containing 2000g of loam-sandy soil) were inoculated with J2 (2000, 3000, 4000 per pot) of nematode and/or microsclerotia (20000 per pot) of fungus according to the treatments for the cultivars. Pots were arranged on glasshouse benches with a temperature of 25-27°C, and under natural light. Quantitative activities of Soluble Peroxidase (SPOX) and ionically Cell-Wall-bound Peroxidase (CWPOX) were evaluated using guaiacol, as a substrate, and according to change in absorbance of the reaction mixture at 470 nm per minute per milligram of total protein on days 1, 10, 20 and 30 after inoculation. Results showed that SPOX and CWPOX activity increased in seedlings inoculated with only fungus reaching its maximum level at the 30th day in Koroneiki. The enzymes activity showed a significant increase in treatments inoculated with nematode and fungus until 20 days after inoculation and afterwards decrease until 30 days after inoculation as compared with the fungus alone treatments (p ≤ 0.05). Results indicated the nematode was able to limit induction of SPOX and CWPOX even against fungus.

The chemical compound DL-β-Amino-n-Butyric Acid (BABA) has been introduced as a promoter of induction of resistance in plants against plant pathogens. In this study, promotion of induction of some such defense compounds as peroxidase and polyphenol oxidase enzymes through DL-β-amino-n-butyric acid application against root knot nematode (Meloidogyne javanica) was investigated in cucumber. Results indicated that inoculation of cucumber by the nematode infected the plant's roots through BABA, stimulated peroxidase activity in root tissues one day after inoculation, enzyme activity reaching its maximum level in four days past nematode inoculation. Induction of polyphenol oxidase activity also showed significant difference in BABA treated plants in comparison control and with healthy plant's reaching its maximum level in four day after nematode inoculation. Native PAGE (Polyacrylamide Gel Electrophoresis) analysis indicated that the peroxidase isozymes in cucumber roots induced by BABA were different from those in roots infected with nematode. In BABA plus nematode inoculated plants, two isozymes (Rf= 0.31 and Rf= 0.34) were induced which were more condense in comparison with control (only inoculated with nematode).

Rice is among the most important food crops throughout the world. Hybrid rice benefits from about a 30 percent yield advantage over the conventional pure line varieties and this phenomenon can play an important role in fighting world hunger. To diminish the damage of rice pests, development of insect pest management strategies in hybrid rice is necessary. This study was aimed at evaluating the grain yield and stem borer infestation of hybrid rice (Daylam) grown in two planting times and when different control methods employed. The study is expected to provide the suitable needed information for implementing IPM in Mazandaran province. A randomized complete block design, involving four treatments (biological, biological-mechanical, chemical vs. control) and of three replications was adopted. To estimate the pest damage, dead hearts and whiteheads were respectively taken from central parts in each plot at the vegetative and reproductive stages respectively. Analysis were made using Duncan's multiple range test to separate means. Results indicated the least average contamination occurring in biological- mechanical control method at either one of the vegetative or reproductive stages. The least overall average contamination occurred in chemical and biological-mechanical control method. In a second trial, percentage of contamination in early planting time treatment was reported as lower than that in late planting time. Therefore, early planting is an effective measure to be taken for decreasing the damage of striped stem borer in Daylam rice in Mazandaran province.

Alfalfa (Medicago sativa) is among a few of the most important forage crops for the growth of which Hamedan province benefits from a suitable climate. Alfalfa bacterial wilt disease induced by Clavibacter michiganensis subsp. insidiosus cause substantial economic crop losses. This disease has not been so far investigated extensively in Hamedan province. In the course of this survey affected plant samples were collected from alfalfa growing areas in Hamedan province, and causative bacterial agents isolated. Pathogenicity of the strains was confirmed on plants and their phenotypic features characterized as based on standard bacteriological methods. Bacterial strains induced symptoms on tested alfalfa plants after a passage of 40 days. Pathogenic strains were identified as C. m. subsp. insidiosus. Effect of the strains on shoot and root length, wet and dry weight of the plant parts were evaluated in a randomized design in four replicates. The obtained data were analyzed through SAS software. Results indicated that all the tested strains reduced the above-mentioned traits showing significant differences in this regard. To determine of the genetic diversity among the tested strains Opa-03, Opa-15, Opb-10, Opb-15, Opb-18, Opb-20, Ope-01, Ope-02, Ope-03, Ope-04, Ope-07, Ope-14, Ope-16 and Ope-19 primers were picked up and clustering was performed. Results revealed four clusters comparised of: AH7, AH8, AH9 and AH6 strains (first cluster), Ah15 and AH2 strains (second cluster), AH10, AH11, AH12 and AH14 strains (third), and AH1 and AH3 (fourth cluster). EcoR1 digestion of the amplified fragment using CIRS-1 and CIRS-2 primers revealed two groups of strains. All the obtained data revealed genetic diversity among the C. m. subsp. insidiosus strains collected from Hamedan province.

Some possible effects of exogenous application of Salicylic Acid (SA) and Bacillus subtilis on infection potential of root and stem rot disease and on induction of resistance against this pathogen were investigated in laboratory and as well under greenhouse conditions. In vitro, the effectiveness of SA concentrations (2-8mM) on growth of B. subtilis was observed as negative, while SA application completely inhibited the mycelial development of F. oxysporum f. sp. radicis-cucumerinum at concentrations exceeding 5mM. In pot experiments, application of SA (3, 5 and 7 mM) and B. subtilis post or/ and prior to inoculation with the pathogen demonestrated that disease index was reduced in cucumber plants treated by SA as a plant defense elicitor and inoculated with B. subtilis as a biocontrol agent, prior to fungal infection causing increase in some growth factors as compared with the infected control. Application of 7 mM of SA (foliar spray) and B. subtilis (soil drench), prior to fungal infection reduced the extent of fungal infection and induced plant growth as compared with control and with other treatments. An evaluation of phenylalanine ammonia lyase activity showed that, combined application of SA and B. subtilis significantly increased the activity of this enzyme as compared with SA and B. subtilis application alone and with control. Maximum level of phenylalanine ammonia lyase activity was observed on the 6th day after elicitor's application. In summary, results suggest that application of SA as a chemical elicitor and B. subtilis as a biocontrol agent plus plant growth promoter, can be a useful and promising measure for a control of such phytopathogens as F. oxysporum f. sp. radicis-cucumerinum.

Entomoparasitic Nematodes (EPN) are among the very important pest control agents. Among the related groups within these nematodes, two families namely Steinernematidae and Heterorhabditidae are more important than the others. Due to commercial application of these agents in pest control, extensive research has been carried out to identify the different species of these nematodes throughout the world. During a survery on the occurrence of EPN in Mashhad, a population of Heterorhabditis genus was isolated from the soil. Based upon morphological and morphometric traits, the isolated species was determined as H. bacteriophora. The ITS rDNA region in the nematode was amplified and sequenced. In a phylogenetic analysis as based on MP and NJ methods, this isolate stood with other populations of H. bacteriophora. The symbiotic bacterium associated with this nematode was characterized based on phenetic data as well as on 16S rDNA sequence. The results revealed this bacterium as Photorhabdus luminescens ssp. laumondii. This presents the first comprehensive repot of this bacto-helminthic complex in Iran.