فصلنامه بیماریهای گیاهی
سال پنجاه و پنجم شماره 1 (بهار 1398)

Host plant cell wall is the main mechanical barrier to penetration and migration by endoparasitic nematodes. In order to degradation of plant cell walls, the nematodes secrete various types of cell wall degrading enzymes. β‐1, 4‐endoglucanase was the first known cell wall degrading enzyme. In this research, the enzyme activites were studied in three species; Meloidogyne incognita (Kofoid & White1919)Chitwood, 1949, Pratylenchus loosi Loof, 1960 and Aphelenchoides besseyi Christie, 1942. Initially, sampling, identification, purification and multiplication of the nematodes were performed and after extraction of their DNA, the encoding β - 1, 4 endoglucanase gene was detected in the nematodes  by using pairs of specific degenerate primers in polymerase chain reaction. After extraction of total RNA from three species of nematodes, cDNA was synthesized and determination of cDNA goodness was carried out by a reference gene. Gene expression of the β -1,4 endoglucaase was done for all samples. The results showed that the gene encoding β -1 ,4 endoglucanase had the highest activity and expression in the root- knot nematode (M. incognita). Tea root lesion nematode (P. loosi) and Rice white tip nematode (A. besseyi) were classified in  next  step, respectively.

Bakanae disease and Foot rot of rice is one of the rice seed-borne diseases that observed from nursery to paddy field. Most important symptoms of disease is production of elongated and abnormal seedlings from contaminated seeds on the effect of Gibberellic acid in nursery and field. This investigation, effect of Gibberellic acid produced by Gibberella fujikuroi evaluated on the increase of stem and root growth and mortality of Hashemi cultivar seeds. Hashemi’s cultivar seeds inoculated by Gibberella fujikuroi conidial suspension isolated from infected seedlings from rice fields of Guilan province. Rate of increase of growth by measuring of stem and root length and number of dead seeds once every five days and amount of Gibberellic acid produced by Gibberella fujikuroi isolates was determined using High Performance Liquid Chromatography (HPLC). Results of quantitative determination of Gibberellic acid showed that all isolates were studied have ability to produce this hormone. There were not found any significant correlation between the amount of production of Gibberellin hormone in pathogen and increase of stem and root length from germination of inoculated seeds of Hashemi cultivar and also number of dead seeds.

Barley is one of the most important cereals in the world after corn, wheat and rice. Powdery mildew is one of the important fungal parasites of the plants which cover their above ground parts with the formation of a white fungal vegetative organs causing yellowing, drying and reducing the quantitative and qualitative traits of the field crops, fruit trees and ornamental plants. In order to locating the powdery mildew resistance QTLs in the F3 generation derived from the cross Badia and Kavir cultivars, a population consisted of 104 families was cultivated in 2017 at Gonbad-e-Kavos University. To provide a genetic map, several groups of markers including of 28 SSR, 9 ISSR, 3 IRAP and 5 iPBS (93 alleles) that were related to barley chromosomes used. Genetic maps were covered 617.5 cM and the mean gap between flanking markers was 5.41 cM. Four QTLs were detected on chromosomes 3, 4 and 6 to powdery mildew resistance. The results of this study led to a new QTL trace for the desired trait. The QTL qSPM-4b with LOD=3.26 that was between the two ISSR13-1 and ISSR16-4 markers, controlled 13/.6 of the phenotypic variations of the respective attribute with an incremental decrease of 0.12. The QTLs detected could be used in breeding programs to increase the resistant sources against the disease and to increase the yield of the new produced cultivars. The results of the current research can be used in breeding programs after determining the validity of markers.

Non-pathogenic Pseudomonadsare the main biocontrol agents that are used in plant disease management. In this study, effects of four strains of Pseudomonas fluorescens (Pf1, Pf2, Pf3, Pf4), and P. putida (P13) were studied for their ability to elicit induced systemic resistance against diseases caused by Botrytis cinerea and Alternaria alternata on tomato (Super Majar and Queen cvs) and Arabidopsis. The ability of biocontrol agents to colonize roots of tomato cultivars and Arabidopsis and induce resistance was shown to be highest for P13, and Pf3 strains and P13, and Pf3 were able to reduce the average lesion diameter of diseased leaves to 30% and 25%, compared to that of control, respectively. The ability of biocontrol agents for direct control the pathogens was also evaluated. The results revealed that P. putida (P13) was the best biocontrol agent as measured by the reduction in disease severity. P13 reduced the average lesion diameter of diseased tomato leaves by about 45%. Finally, Pf3 and P13 were identified as more powerful strains. Moreover, these strains could induce some level of resistance against various pathogens and they had the ability to enhance plant growth.

Twenty-one bacterial strains were isolated from wheat showing spike blight disease symptoms in southern Kerman province. Differential phenotypic features of the strains were studied using common bacteriological methods. Molecular identification of the isolates was studied by amplification and partial sequence analyses of 16S rRNA, gyrB and recA genes. PCR assays using specific primer sets amplified 700 and 558 bp amplicons of 16S rRNA and gyrB genes from all strains, respectively. PCR assay with specific primer set for recA gene amplified a 600 bp fragment from Rathayibacter iranicus, but not from R. tritici strain and the isolated strains from wheat in southern Kerman province, as well. Sequences of the amplicons obtained from each gene locus were identical to those sequences for R. tritici reference strains as well as the R. tritici strains available in GenBank. Results obtained from phenotypic tests and analyses of the data from amplification and sequencing of the studied gene loci indicated that R. tritici is the causal agent of wheat spike blight disease in southern Kerman province. To the best of our knowledge, this is the first report of R. tritici on wheat in Kerman province.

Erwinia amylovora is a gram-negative bacterium that causes fire blight, an important disease in pome fruit trees. The aim of this study was to generate a transposon mutant library using mini-Tn5 lacZ1, in an Erwinia amylovora native isolate for identification of some virulence associated genes in this strain. Among 1500 mutated colonies, two colonies which indicated less virulence on immature pear fruits were selected for further analysis. The DNA sequencing results of the two selected mutants showed a transposon insertion in an araC family transcriptional regulator gene and an insertion in nonribosomal peptide synthetase (NRPS). This study demonstrated that the araC transcriptional regulator and the NRPS genes are required for full virulence of E. amylovora. More studies are necessary to identify the role of these genes in physiology and virulence of E. amylovora.

Root-knot nematodes(Meloidogyne spp.) are the most important parasitic nematodes infecting cucurbits plants. These nematodes cause 5% loss in crop production and also provide favorable conditions for co-infection with other plant pathogens. Trichoderma harzianum Rifai is a potential bio-control agents for controlling the plant pathogens, including plant parasitic nematodes. In order to find the effective isolate/s of T. harzianum for management of root-knot nematodes on cucumber, a suspension of 107 spores/ml of four isolates of T. harzianum with 36, 39, 42 and 43 codes was evaluated by soil drenching method. Twenty four hours after soil drenching of fungus isolates, four leaf stage seedlings of cucumber (cv. Super Dominus) were inoculated with 2000 eggs and second stage juveniles of Meloidogyne javanica and kept for two months in the greenhouse and the experiment was conducted as a completely randomized design with four replicates. The results showed that the isolate Th42 was the most effective isolate on reducing the biological activity of nematode as compared to other treatments. The isolate Th42 reduced the number of eggs, galls, egg masses and reproductive factor by 69.2, 81.8, 88.6 and 70%, respectively, as compared to control treatment.

Broomrape species (Orobanche spp.), obligate parasitic plants, are considered as the limiting factors for cultivation of several important agricultural crops which cause damage and crop lost in dicotyledons such as tobacco, tomato, eggplant, potato, sunflower and carrot (Barker et al., 1996; Sauerborn, 1991). Although several traditional methods have been used to control broomrapes on different crops, none has proved to be effective (Amsellem et al., 2001). On the other hand effective methods such as soil fumigation are expensive and also have environmental risks (Foy et al., 1989). Biological control is an alternative control method and a number of promising fungal isolates have been reported all over the world with satisfying level of control on these pathogens (Boari and Vurro, 2003). Fusarium oxysporum, F. solani, Rhizoctonia solani and Urocystis orobanches have been reported from broomrape in Iran (Ershad, 2009). Rostami and co-workers (2015) isolated F. proliferatum, F. torulosum and F. circinatum. Also F. chlamydosporum, F.solani, F. semitectum, F. oxysporum, F. reticulatum, F. pallindoroseum, F. diversisporum and F. virguliform were reported fromEgyptian broomrape, O. aegyptiaca (Darvishnia et al., 2013).In order to study fungi associated with broomrape, samples of O.ramosa with wilt and/or rot symptoms in their stem and crown were collected from tomato fields in Firoozabad and gharegheshlagh villages in Kosar city, Ardabil province. Pieces of stems were first washed with running tap water, sterilized in 1% sodium hypochlorite for 2 min, and rinsed with sterile distilled water. The fragments were placed on potato dextrose agar and incubated at 25 °C and observed daily for fungal growth. The fungal isolates were purified and stored. Finally a number of 90 isolates were obtained of which 46 isolates were identified based on their morphological and microscopic characteristics on PDA and CLA (for Fusarium species) (Leslie and Summerell, 2006). Also genomic DNA of a representative isolate for each morphologically identified species was extracted (Liu et al., 2000) and their internal transcribed spacers of ribosomal gene (ITS1 + 5.8S + ITS2) was amplified using the primers ITS1 and ITS4 (White et al. 1990). PCR products were sent to Macrogen Inc. (South Korea) for purifying and sequencing. The sequence of amplicons was compared with sequences from NCBI using BLAST. Isolates belonging to the species of dominant genera were inoculated on broomrape tubercles in transparent plastic bags containing tomato seedlings. After three weeks the symptoms were assessed based on a visual score as follow: 0: nonpathogenic; 1:slight symptoms on tubercles, such as browning and slackening of growth rate; 2: inhibition of growth and tubercle browning and necrosis; 3: quick and complete necrosis of tubercles, loss of consistency. Three replicates were used for each species. Based on the morphological and molecular characteristics of 46 isolates, the isolates belonged to three genera Fusarium (frequency: 3/41%), Macrophomina (frequency: 4/54%) and Aspergillus (frequency: 3.4%). This is the first report of the occurrence of Aspergillusochraceus and F. fujikuroi on broomrape in the world. Also before this research there is no report on the occurrence of Macrophomina phaseolina, F.acuminatumand F.equiseti on broomrape in Iran. Based on the results of pathogenicity experiment, all tested Fusariumspecies and M. phaseolina caused disease symptoms on broomrape tubercles. Among the species, F. acuminatum, F. fujikuroi and F.proliferatum caused the most disease severity on tubercles.