نشریه مهار زیستی در گیاه پزشکی
سال هشتم شماره 1 (بهار و تابستان 1399)

Crown gall is one of the most damaging bacterial diseases caused economic damages. Nowadays special attention has been focused on biological control of plant diseases as an alternative to chemical control in terms of the dangers of pesticides. There was no special research focused on the interaction between Bacillus and Agrobacterium until now. The objective of this study was understanding molecular mechanisms between B. subtilis as a biocontrol agent and A. tumefaciens. This information would provide us the opportunity for better management of the disease. In this study tobacco plants (Nicotiana tabacum), IBRC–M10701strains of A. tumefaciens, B. subtilis strain ATCC21332, and Bacillus subtilis OKB105 strain FKR3 were used. miRNAs have an important effect in the auxin signaling pathway and resistance induction. The expression level of two miRNAs, miR167 and miR393 was measured using Real–Time PCR; 1, 3 and 6 days after inoculations of A. tumifaciens and B. subtilis revealed significant differences with the control plants. The results of expression levels demonstrated FKR3 showed a better effect to ATCC21332 in controlling A. tumifaciens. Research showed a positive effect of Bacillus on Agrobacterium. This study indicates the possible use of biological controls such as B. subtillis in controlling of A. tumefaciens.

Increasing need for wood and on the other hand declining wood resources have caused desire for afforestation with fast–growing species such as poplars, but poplar species usually are susceptible to drought. Ectomycorrhizal fungi can improve the water status of host plants and increase plant survival and growth under drought conditions. The precise mechanism of this function is still not clear, but amino acid metabolism has been reported to play an important role in plant tolerance to drought stress. In this study seedlings of Populus caspica were propagated through tissue culture and their symbiosis with Pisolithus arhizus was carried out to evaluate the plantlets resistance to drought in acclimatization process. It was found that establishment of this symbiosis enhanced seedling height, biomass of root, shoot and leaves, photosynthesis, transpiration, stomatal conductance and leaf chlorophyll. Determination of amino acids in ectomycorrhizal and non–ectomycorrhizal roots of P. caspica plantlets (in both drought and irrigation conditions) showed that generally, the concentration of most amino acids increased in ectomycorrhizal root systems, which proved amino acids are the most important assimilation and nitrogen transfer forms in ectomycorrhizal tissue. Significant accumulation of many amino acids such as asparagine, glutamine, glutamic acid, histidine, tyrosine, threonine, alanine and methionine in ectomycorrhizal roots under stress conditions (compared to the ectomycorrhizal roots under irrigation and the control roots) was found, thus it can conclude that tolerance of ectomycorrhizal roots to drought stress is closely associated with their accumulation of free amino acids. Arginine, citrulline and aspartic acid accumulated in non–inoculated P. caspica root systems under irrigation conditions (compared to the mycorrhizal root systems), which confirmed the use of these amino acids in some metabolic pathways in the mycorrhizal root systems. Implications of these changes in the functioning of the ectomycorrhizal symbiosis are discussed.

In this study, three nonpathogenic endophytic bacterial isolates recovered from Fragaria × ananassa stolon and petal and their antagonistic activity was evaluated against Colletotrichum nymphaeae, the causal agent of strawberry anthracnose under in vitro, in vivo, and greenhouse conditions. Bacterial isolates were identified as Bacillus spp. using a combination of phenotypic, biochemical properties and molecular phylogenetic analysis of the 16S rDNA gene sequence. The living cells of bacterial isolates inhibited the mycelial growth of C. nymphaeae using dual culture method and inhibition percentage was evaluated as 41.87, 50, and 54.92 % in MarG2, MarD40 and MarD35, respectively. The volatile compounds (VOCs) produced by bacterial isolates inhibited the mycelial growth of C. nymphaeae and the highest inhibition was observed in MarD35 isolate (24.7%). Moreover, the cell free culture filtrates of bacterial isolates reduced the mycelial growth and conidial germination of pathogen and the highest inhibition was observed in MarG2 (34.96%) on mycelial growth and in MarD35 (52.07%) on conidial germination. The living cells of bacterial isolates decreased anthracnose fruit rot at postharvest and the highest inhibition was observed in MarD40 (85.63%) and MarD35 (81.72%), respectively. Furthermore, disease severity of strawberry anthracnose by bacterial isolates was reduced using drenching soil and plant spraying techniques. The findings of this study showed that bacterial isolates have the ability to prevent the growth of fungal pathogen under in vitro, in vivo and greenhouse conditions.

Phenacoccus solenopsis Tinsley is a polyphagous pest that has become a serious threat to agriculture in the tropical and subtropical regions of the world since the beginning of the twentieth century. Aenasius bambawalei Hayat is an endoparasitoid that parasitizes third instar nymphs of P. solenopsis. In this study, sublethal effects of spirotetramat and chlorpyrifos were investigated on the functional response of the parasitoid on the nymphs of P. solenopsis at 27± 1 ° C, 65± 5% RH, and a photoperiod of 14: 10 (L: D). The densities of 2, 4, 8, 16, 32, and 64 third instar nymphs of P. solenopsis were used in the experiments. The results of logistic regression showed type II functional response in all three treatments including control, spirotetramat, and chlorpyrifos. The search rate (a) of the wasp in control, spirotetramate, and chlorpyrifos treatments were calculated to be 0.2609, 0.1107, and 0.0556 h–1, respectively, which in spirotetramat, and chlorpyrifos were 57% and 78% less than control. Handling time (Th) in control, spirotetramat, and chlorpyrifos treatments were calculated to be 0.0187, 6.3352, and 9.3903 h, respectively. In spirotetramate and chlorpyrifos treatments, handling times were about 26% and 87% more than control. The handling rates of control, spirotetramate, and chlorpyrifos were 4.63, 3.28, and 2.55 host nymphs, respectively; which in spirotetramate and chlorpyrifos it was 31% and 45% less than in control. The results of this study showed that the insecticides of spirotetramate and chlorpyrifos had a negative effect on the functional response behavior of the parasitoid even at low doses and thus could disturb the biological control.

Rice foot rot disease caused by Fusarium fujikuroi, is one of the most important rice diseases in the world and in Iran. Integrated management with different methods is very important for the disease. In this research, to find appropriate fungal antagonistic isolates for the biological control of foot rot, the effect of four isolates of Trichoderma spp. and three isolates of Aspergillus spp. were studied on F. fujikuroi in vitro using dual culture, slide culture, volatile metabolites and non–volatile metabolites. The results of research showed that T. harzianum isolate with 81.81% was the most effective isolate in suppressing the mycelium growth of F. fujikuroi. In the volatile metabolites method, T. harzianum and T. virens isolates were the most effective with 73.33% in suppressing the mycelium growth of the pathogen. In the non–volatile metabolites method, A. awamori, T. viride and T. koningi isolates showed the highest percentage of F. fujikuroi inhibition. In the slide culture method, all isolates of Trichoderma spp. and Aspergillus spp. proved to be effective in inhibition of the mycelia of F. fujikuroi. These fungal isolates were inoculated on the rice plant under greenhouse conditions. All inoculated fungi were able to reduce the severity of the disease in the treated plants. The isolates of T. harzianum and T. virens were the most effective fungi in reducing the disease by reducing the severity by 83% and 79%, respectively. Also, application of all the studied fungi in the greenhouse increased the height, fresh weight and dry weight of shoots and roots in the presence of pathogen compared to the control plants (without pathogen). Analysis of variance and comparison of the mean of the treatments showed a significant difference between the fungi except in the dry weight trait. According to the results obtained from biocontrol studies in laboratory and greenhouse, T. harzianum, T. virens and T. viride isolates were the most effective antagonists in controlling rice foot rot disease.

Rice blast disease, which is caused by Pyricularia oryzae, is one of the most important diseases of rice in the world. In this research, out of 150 infected samples collected from rice farms of Guilan province, 20 fungal isolates and 30 bacterial isolates were collected. The isolated bacteria were belonged to Pseudomonas aeruginosa, Enterobacter sp., Enterobacter cloacaeand Bacillus subtilis. Two methods of dual culture and the production of volatile antifungal metabolites were used in the laboratory in order to study biological control. In the greenhouse trial, rice plants (cv Hashemi) were inoculated with the six bacterial isolates as well as P. oryzaepathogen after 12 days. According to the results of this research, in the dual culture method, P. aeruginosa Y–25–3 with 61.11% and in the volatile metabolite method, B. subtilis W with 60.51% were found to be most effective in suppressing mycelial growth of the pathogenic fungus. All isolates significantly alleviated the severity of the P. oryzae–induced disease in greenhouse, but the most effective was P. aeruginosa, which reduced the disease index by 70.53%. Analysis of variance and comparing the characters average in Duncan test and Least Significant Difference (LSD) and based on a completely randomized design revealed significant differences among the bacteria in terms of mycelial inhibition in the dual culture and volatile metabolite methods. Furthermore, in greenhouse trials there were significant differences in the disease index, plant height and fresh weight among the treatments, whereas the treatments did not exhibit any significant differences in dry weight. Based on the results of biocontrol assays in the laboratory and greenhouse, P. aeruginosa Y–25–3 was recognized as the most effective bacterial isolate in the control of rice blast disease.

Quorum sensing (QS) phenomenon is a cell density-dependent regulatory system that bacteria use to communicate with each other as a multicellular organism. Numerous Gram-negative and Gram-positive bacteria use this signaling system to synchronize genes expression and group behaviors including virulence factor secretion, antibiotic production, competence, biofilm formation, and bioluminescence. In this signaling system, bacterial cell-to-cell communication mediated by sending and sensing small signaling molecules known as autoinducers (AIs). For the prohibition of the quorum-sensing system in the bacterial cell, there is another regulatory system named quorum quenching (QQ). The function of this system is through the secretion of inhibitors (QSIs) and some enzymes (QQ enzymes) which interfere with communication in the quorum-sensing system. Due to the inhibitory potential of the QQ system to disrupt the communication network provided by the QS system, it introduced as a novel strategy to control plant bacterial diseases.

Yeasts are single–celled microorganisms classified as members of the fungi kingdom. This group are very important in biological control, so that several species have been produced and supplied as biocontrol products at the commercial level. In Iran, several types of yeast have been isolated and introduced as biocontrol agent. Therefore, the purpose of this study is to know the yeast biodiversity from agricultural microflora. For this reason, epiphytic yeasts on crop plants and fruit trees were collected from the Northern provinces and some other parts of Iran during 2015–2016. The yeast isolates were recovered on malt agar (MA) medium. All yeast isolates were basically grouped according to the morphological characteristics of colonies. Based on morphological charecterestics such as color and colony form, 24 isolates were selected as reperesentatives for identification. The results of biochemical tests using Simple Matching Coefficient in 87% showed that all isolates are grouped into 14 separate clusters. Twenty–four isolates representatives of 14 groups were selected and analyzed by partial sequencing of ITS1–5.8S–ITS2 region with ITS1 and ITS4 primers. All isolates were identified as Aureobasidium pullulans, Candida membranifaciens, Cystobasidium minutum, Debaryomyces hansenii, Filobasidium magnum, Metschnikowia koreensi, Naganishia albida, Ogataea zsoltii, Papiliotrema flavescens, Rhodotorula glutinis, R. mucilaginosa and Sporobolomyces ruberrimus. Among them, four species i.e. O. zsoltii, M. koreensi, P. flavescens, C. minutum are new records to the Iranian mycoflora. All yeasts were described by macroscopic and microscopic morphological characters including colony color and shape; cell size and shape; and by location of isolation.

Pistachio is an important export product in Iran. This product is constantly threatened by Aspergillus flavus and its aflatoxin. Researchers are now looking to use alternative non-hazardous methods instead of other methods of management of plant diseases, such as the use of chemical pesticides. Plant extracts as an environmentally friendly method can be a good alternative to chemicals. In this study, the effect of plant extracts including lavender (Lavandula stoechas), thyme (Thymus vulgaris), fennel (Foeniculum vulgare) and savory (Satureja hortensis) on the amount of degradation and aflatoxin production in vitro, by paper disk methods and mixing the extract with the culture medium, as well as in the storage conditions and on pistachio were investigated. The results showed that using of 2000 mg/L methanol extract reduced the growth of pathogenic fungus by 65.63%. In the tests of using the extracts on the paper disc as well as mixing the extracts with the culture medium, aqueous extract of lavender with a concentration of 2000 μg/ml prevented the growth of pathogen by 39.31 and 55.96%, respectively, and thyme extract with a concentration of 2000 μg/ml inhibited the fungal growth by 29.33% and 39.54%, respectively. These extracts had the highest effect on the pathogen growth. When the extracts were applied directly to aflatoxin, the results showed that the concentration of 4000 mg/L lavender prevented the aflatoxin production in the culture medium by 91.18%. Finally, aqueous extract of lavender at 4000 mg/L showed the highest effect (76.95% compared to control) in reducing the amount of spores produced by pathogenic fungi on pistachio fruit. The results of aflatoxin content in pistachio showed that both studied plants (lavender and thyme) at concentrations of 2000 and 4000 mg/L significantly reduced the amount of aflatoxin produced. Due to the remarkable and good effect of plant extracts on pistachio fruit under both in vitro and in the storage, the studied extracts, especially lavender and thyme, are natural compounds with high fungicidal effect and environmentally friendly effects.

Plant pathogens cause great damage on crops during harvest and post–harvest stages. The use of synthetic chemical fungicides is inevitable and causes irreparable damage to the environment and human health. Replacing chemical fungicides with beneficial biological agents can be effective in reducing these risks and damages. Among biological agents, yeasts are promising alternatives to chemical plant protection compounds due to their widespread distribution, bioavailability, environmental friendliness, and safety for human. Over the past few decades, the study of biocontrol mechanisms of yeasts against plant diseases has been extensively studied. Here, the fundamental researches on the mechanisms (e.g. competition, enzyme secretion, toxin production, volatiles compounds, mycoparasitism, and induction of resistance) of biocontrol yeasts as plant protection agents are reviewed.  It also provides an overview of the commercialization process and registered yeast–based bioproducts. In general, this study shows the lack of fundamental studies on the biocontrol mechanisms of yeasts and registered yeast agents. Therefore, identifying the biocontrol mechanisms of yeasts in more detail is still one of the research fields and better understanding of them can pave the way for the development of yeast–based commercial products for plant protection.

Identification of hemocytes is one of the basic studies of circulatory system physiology. Blood cells, as the first defense barrier to insect cellular immunity, participate in blocking the foreign particles in the form of phagocytosis and nodules. In the present study, homocytes of adults of Blepharopsis mendica were identified after staining with Gimsa and using a light microscope with a magnification of 40. In the hemolymph of this predatory insect, five types of homocytes were observed, including prohemocytes, granulocytes, plasmotocytes, oenocytoids, and spherulocytes. The most abundant blood cells in the nymphs and adults of this mantis were spherulocytes and plasmotocytes, respectively. The lowest abundance of cells belonged to prohemocytes. Oenocytoids were the largest cells. Oenocytoids had a large nucleus that extended almost to the side of the cell membrane and the cytoplasmic surface of these cells was granular. Prohemocytes were the smallest cells and observed with the distinct central nucleus. Plasmotocytes were observed as spindles, and the common polymorphic profile of most insects hemolymph was not observed in hemolymph of B. mendica. Granulocytes size were small to medium but were larger than prohemocytes with granule–like granules in cytoplasmic surface. Spherulocytes are medium to large, have a central nucleus, and numerous spherul occupy the cytoplasm of these cells. Adult body weight and blood volume were higher than the nymphs of this mantis. So, it is clear that there are higher populations of hemocytes in adults in comparison nymphs. The identification of mantis homocytes as a large group of pest predators is being investigated for the first time and can be used in the study of its immune features.

Pectobacterium carotovorum is the causative agent of potato soft rot. The aim of this study was to find an effective and safe solution to reduce the damage of this disease. The antibacterial effect of ethanolic extract of two medicinal plants i.e. thyme and black cumin on the pathogenic factors of P. carotovorum was investigated in a completely randomized design and the factors of inhibition zone diameter, inhibition percentage, biofilm, hydrophobic effect, adhesion on the potato tuber and motility of bacteria were measured and analyzed. The results showed that the maximum diameter of the inhibition zone due to the use of thyme extract was 9.66 mm. Also, thyme plant extract had a significant effect on the inhibitory percentage of P. carotovorum in the potato tuber by 87.46% and reduced the symptoms of the disease on the potato tuber. Effects of thyme and black cumin extracts on the other pathogenic factors were the same and both extracts showed statistically significant differences with the control. It seems that thyme extract had more antibacterial properties and because of inhibition of motility of bacteria caused significant control of the disease on the potato tubers. According to the results of this study, the use of effective compounds of thyme extract to control soft rot disease of potato tuber, especially during storage is recommended.