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We present the data corresponding to the ultrastructural characteristics of Paenibacillus polymyxa isolates and control efficacy of P. polymyxa ShX301 for controlling Verticillium wilt of cotton, isolated in experimental fields at the Sanyuan Agricultural Experiment Station of North-West Agriculture and Forestry University, Sanyuan county, Shaanxi province, China. Ultrastructural characteristics of P. polymyxa isolates made using technique of transmission electron microscopy. A strain ShX301 has a broad-spectrum antifungal activity against V. dahliae and other plant pathogens and has been used for in vitro experiments for controlling this disease in greenhouse, ''Biocontrol potential of Paenibacillus polymyxa against Verticillium dahliae infecting cotton plants'' [1].

Verticillium wilt of cotton is an economically important disease caused by V. dahliae throughout the world. To control this disease, five bacterial strains from cotton-growing fields were screened for their antagonistic activity against V. dahliae. Interestingly, they were all identified as Paenibacillus polymyxa by testing morphological and ultrastructural characteristics as well as 16S rRNA sequences and fatty acid composition analysis. The observation of sporulation process revealed that the structure of the endospore coat was composed of three layers including outer, middle and inner spore coats. Among five strains, ShX301 showed highest antagonistic activity against spore germination and mycelial growth of V. dahliae. In addition, in vitro experiments showed that ShX301 had a broad-spectrum antifungal activity against other plant pathogens. The experiments for controlling Verticillium wilt of cotton in greenhouse demonstrated that inoculation by strain ShX301 reduced disease incidence and severity by 71.4% and 40.3%, respectively. Moreover, ShX301 significantly promoted the growth of cotton seedling. Assessment of plant growth promotion showed that inoculation by ShX301 promoted cotton plant growth enhancing aboveground seedling biomass by 45.5% as well as underground root length and biomass by 38.0% and 38.5%, respectively. This is the first report of P. polymyxa application to control Verticillium wilt of cotton and promoting cotton seedling growth. This study revealed that P. polymyxa ShX301 had high potential to be used as biocontrol agent and/or plant growth-promoting bacterium in agriculture.

This research was carried out to assess inhibitory effect of UV-C (0.88 kJ m−2) against four isolates of Sclerotinia sclerotiorum, causal agent of carrot rot, in vitro and in vivo. A bank of seven germicidal tubes was used to produce UV-C radiation with peak emission at 254 nm. The carrot roots were subjected to UV-C irradiation for five min. The tubes were located 40 cm above the roots. In order to be exposed to UV radiation on all sides, the carrot roots were rotated five times at the interval of one min. Based on the results, UV radiation was able to significantly increase levels of pathogenesis-related proteins (chitinases, peroxidases, β-1,3-glucanases) as well as polyphenoloxidase, phenylalanine ammonia-lyase and 6-methoxymellein (6-m) in carrot roots. The levels of enzymes and 6-m were found to be higher in the whole treated carrots than half treated carrots in most of treatments. Despite increase of systemic acquired resistance (SAR) related enzymes in half treated carrots, the results of in vivo tests showed that UV-C irradiation can not reduce severity of carrot rot after 15 d. In addition, UV-C radiation for 5 min markedly decreased myceliogenic and carpogenic germination of sclerotia in four isolates. This study showed that UV-C cannot be considered as abiotic inducer of SAR.

A new species, Scopulariopsis gossypii, was found to be present in the vascular bundles of cotton plants (Gossypium hirsutum) infected by Verticillium dahliae which is an economically important pathogen in Hangzhou, China. The fungus was only present in the diseased plants, but it never became isolated from the healthy plants. Scanning electron microscopy showed that the fungus was present in parenchyma cells enclosing vessels in dark brown vascular tissues of stems, and produced asexual conidia within the tissues. Phylogenetic analysis of combined nuclear ribosomal D1/D2 region of the 28S rDNA as well as translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB) gene showed that S. gossypii represents an undescribed species of Scopulariopsis, Microascaceae. In this study, characteristics of sexual and asexual stages of the fungus were described, illustrated and compared with similar taxa. In addition, the molecular and morphological analyses indicated that S. gossypii was a distinct species of Scopulariopsis. The pathogenicity tests proved by inoculation of wounded roots confirmed that S. gossypii was an opportunistic pathogen causing leaf interveinal chlorosis and vascular browning of cotton plants. However S. gossypii did not infect host with undamaged roots. Moreover, coinoculation with S. gossypii and V. dahliae significantly increased disease severity.

Potato white mold caused by Sclerotinia sclerotiorum is an important plant disease occurring in many potato-producing areas worldwide. This disease causes considerable yield losses in a large number of potato fields of Hamedan province, Iran. Although several non-chemical methods have been suggested against the pathogen, application of chemicals is considered a major method employed by the growers in Hamedan fields leading to considerable pollution of underground water. Therefore, it is necessary to find techniques to help growers make a more timely decision for chemical application. In this research, a pair of PCR primers was designed according to the nucleotide sequence of a region of DNA amplified by the primer M13. This primer set amplified a 352-bp fragment from all isolates of S. sclerotiorum collected from Hamedan, Iran, and Hangzhou, China. However, this situation was not observed in other tested fungi. A real-time PCR assay was conducted to detect early infection of the pathogen on potato petals. This real-time PCR assay is suggested for growers to make timely decisions on chemical application.

Phytophthora capsici is a phytopathogen that causes a destructive pepper blight that is extremely difficult to control. Using a fungicide application against the disease is costly and relatively ineffective and there is also a huge environmental concern about the use of such chemicals. The genus Trichoderma has been known to have a potential biocontrol issue. In this paper we investigate the mechanism for causing the infection of T. asperellum against P. capsici. Trichoderma sp. (isolate CGMCC 6422) was developed to have a strong antagonistic action against hyphae of P. capsici through screening tests. The strain was identified as T. asperellum through using a combination of morphological characteristics and molecular data. T. asperellum was able to collapse the mycelium of the colonies of the pathogen through dual culture tests by breaking down the pathogenic hyphae into fragments. The scanning electron microscope showed that the hyphae of T. asperellum surrounded and penetrated the pathogens hyphae, resulting in hyphal collapse. The results show that seven days after inoculation, the hyphae of the pathogen were completely degraded in a dual culture. T. asperellum was also able to enter the P. capsici oospores through using oogonia and then developed hyphae and produced conidia, leading to the disintegration of the oogonia and oospores. Seven days after inoculation, an average 10.8% of the oospores were infected, but at this stage, the structures of oospores were still intact. Subsequently, the number of infected oospores increased and the oospores started to collapse. Forty-two days after inoculation, almost all the oospores were infected, with 9.3% of the structures of the oospores being intact and 90.7% of the oospores having collapsed.

This study was conducted to determine the inhibitory effect of E-cinnamaldehyde (EC) against causal agent of storage carrot rot, Sclerotinia sclerotiorum, under in vivo and in vitro conditions. Based on the results, EC was able to completely inhibit mycelial growth of three isolates (P>0.05) in both volatile and contact phases after 6days at the concentrations 200μl and 1μl/ml, respectively. In addition, EC at concentrations 1 and 10μl/ml completely inhibited carpogenic germination of three isolates. The results of in vivo trials showed that EC at the concentration of 10μl/ml was able to control the disease caused by isolates 1 and 3. However the disease caused by isolate 2 was inhibited with the concentration of 20μl/ml. In enzyme analyses, the activity of polyphenoloxidase and peroxidase did not change in the inoculated carrots after application of EC. Furthermore, the level of phenylalanine ammonia lyase decreased. These results indicated that EC does not have any potential to be considered as resistance inducers against sclerotinia carrot rot.

Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H2O2 and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.

This research was carried out to assess the effect of E-cinnamaldehyde (EC) against Sclerotinia sclerotiorum , causal agent of potato white mold, under invivo and invitro conditions. Based on the results, EC was able to completely inhibit ( P −1 protein in sterilized distilled water and ethyl acetate, respectively, it was recorded as 78.9 and 86.4 units mg −1 protein for cinnamon extract and EC, respectively.

The in vitro and in vivo experiments were conducted to evaluate antifungal activity of crude extracts derived from neem (Azadirachta indica) leaves and ginger (Zingiber officinale) rhizomes against three isolates of Sclerotinia sclerotiorum, the causal agent of storage carrot (Daucus carota) rot. Ethyl acetate (EA) and ethanol were used as different solvents. Although plant extracts significantly reduced the mycelial growth of three isolates at volatile phase, highest inhibitory efficacy was observed at contact phase. Furthermore, all crude extracts were able to reduce carpogenic germination of sclerotia at concentrations 2 g/l. The isolate 2 was found to be more inhibited in in vitro tests. At volatile phase, EA and ethanol extracts of ginger meaningfully reduced myceliogenic germination of sclerotia in three isolates. However, all plant extracts decreased the myceliogenic germination of sclerotia at contact phase at the concentration 10 g/l. The results of in vivo tests showed that all plant extracts are able to decrease disease severity of carrot rot at the concentration 2 g/l. In addition, the enzymatic analyses of phenylalanine ammonia layse, polyphenoloxidase and peroxidase in inoculated carrots after application of plant crude extracts indicated that neem extracts can be considered as resistance inducers against Sclerotinia carrot rot.

The objective of this study was to assess the antifungal potential of plant crude extracts derived from cinnamon and rosemary against three isolates of Sclerotinia sclerotiorum under in vitro and in vivo conditions. The crude extracts were obtained using two different solvents including ethyl acetate (EA) and ethanol. The results showed that crude extracts of cinnamon are able to reduce mycelial growth of isolate 2 at volatile and contact phase by 35.4% and 78.2%, respectively. Furthermore, sclerotial myceliogenic at contact phase and carpogenic germination of isolate 2 were inhibited by 94.3% and 68.1%, respectively. In general, rosemary extracts showed less inhibitory efficacy than cinnamon. As the most effective treatment, EA extract of cinnamon was analysed using Gas Chromatography/Mass Spectrometry. The results showed the presence of 33 components and the major constituents were E-cinnamaldehyde (66.4%) followed by Alpha-Muurolene (4.86%), Alpha-Copaene (4.73%) and 2H-1-Benzopyran-2-one (3.72%). The enzyme analysis showed that the activity of phenylalanine ammonia lyase, polyphenoloxidase and peroxidase decrease in the inoculated carrots after application of plant crude extracts indicating that they cannot be considered as resistance inducers against Sclerotinia carrot rot. In conclusion, cinnamon extract was found to be more effective against the pathogen. Although crude extracts of cinnamon and rosemary were able to reduce severity of carrot rot during storage, EA extract of cinnamon (2 g L−1) was found to have practically significant effect against the disease.

Ustilago esculenta infects Zizania latifolia plants and induces swelling on their upper culms. As an edible aquatic vegetable, these swellings are called jiaobai (white bamboo). The process of fungal development in Huijiao (grey bamboo) plants from seedlings to upper culm swelling was investigated using knife cut tissues, light and electron microscopy. All Huijiao plants were relatively taller than all jiaobai plants and in knife-cut sections sori development differed after culm swelling in Huijiao plants. Light microscope examination show that hyphae were both intracellular and intercellular, forming hyphal aggregations, and were more abundant in nodes than in internodes. Rapid increase of hyphal aggregations occurred 15 days before culm swelling, causing the death of abundant host cells, and hyphal aggregations differentiated into sori initiates. Ultrastructural observation showed that intracellular hyphae at the interface of fungus-host interaction were encased in a sheath with outer electron-opaque and inner electron-dense layer. Scale-like material and membrane-bounded vesicles were often present in an outer electron-opaque matrix of the sheath. Vacuoles, lipid globules (LGs) and multivesicular bodies (MVBs) were occasionally seen in these fungal cells. In summer, abundant electron-dense MVBs, LGs and fewer vacuoles containing MVBs were present in a fungal cell. As the temperature decreased, some MVBs became electron-opaque, some internal vesicles were also electron-opaque, and subsequently, the MVBs disappeared, leaving irregular remnant materials in vacuoles. During the rapid increase in hyphal aggregations, the irregular remnants of material within the vacuoles disappeared and LGs were rare. At the beginning of sori development, LGs once again became abundant in the cells of hyphal aggregation. The process of sorus and teliospore development was observed systematically and relationships between each developmental stage examined from knife cut tissues, and cytological and ultrastructural sections. Sorus development occurred with the commencement of upper culm swelling and differed in the same tissues. Teliospore development was also uneven in a sorus. The inner region of a sorus developed earlier than the outer part.

This study was conducted to evaluate antifungal activity of three chemically different chitosans named as A, B and C at different concentrations against three isolates of Sclerotinia sclerotiorum, the causal agent of storage carrot rot, by in vitro and in vivo tests. In addition, potential of SAR (systemic acquired resistance) induced by the chitosans and acetyl salicylic acid (ASA) was assessed against the disease. The degree of N-deacetylation (DN) and the molecular weight of chitosans A, B and C were 85%–1129 kDa, 95%–521 kDa and 75%–607 kDa, respectively. The concentrations 2 g/L and 5 g/L of all chitosans were significantly able to decrease mycelial growth, sclerotia formation and carpogenic germination of the pathogen. Four hours and three days before inoculation with the pathogen, application of chitosans and ASA reduced severity of carrot rot in all isolates. However, mycelial growth and sclerotia formation of isolate 2 was more reduced than other isolates. In accordance with in vitro tests, chitosan B showed the highest inhibitory efficacy against the disease. The fungicidal effect of chitosan increased when the DN decreased and there was a negative correlation between the mycelial growth inhibition and the molecular weight of chitosans. The enzyme analysis showed that the activity of phenylalanine ammonia layse, polyphenoloxidase and peroxidase increase in the inoculated carrots after application of different chitosans and ASA.

State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China (Received on November 30, 2011; Revised on May 2, 2012; Accepted on May 14, 2012)Sclerotinia sclerotiorum is a serious pathogen whichcauses yield loss in many dicotyledonous crops includ-ing potato. The objective of this study was to assess thepotential of biofumigation using three Brassica cropsincluding Brassica napus, B. juncea and B. campestrisagainst potato stem rot caused by S. sclerotiorum by invitro tests. Both macerated and irradiated dried tissueswere able to reduce radial growth and sclerotiaformation of five pathogen isolates on PDA, but mace-rated live tissues were more effective. Compared withother tested crops, B. juncea showed more inhibitoryeffect against the pathogen. The volatile compoundsproduced from macerated tissues were identified usinga gas chromatograph-mass spectrometer. The mainidentified compounds were methyl, allyl and butyl iso-thiocyanates. Different concentrations of these com-pounds inhibited mycelial growth of the pathogen invitro when applied as the vapor of pure chemicals. Anegative relationship was observed between chemicalsconcentrations and growth inhibition percentage. In thisstudy, it became clear that the tissues of local Brassicacrops release glucosinolates and have a good potential tobe used against the pathogen in field examinations. Keywords : dual culture, glucosinolates, ion trap detector,volatile compoundsPotato white mold, also known as stem rot, is a damagingdisease caused by the fungus Sclerotinia sclerotiorum. Thisdisease usually occurs in a large number of potato fieldsin Hamadan, Iran (Ojaghian, 2009) resulting in reducedyield and tuber quality. Application of fungicides, mainlyiprodione and dichloran, is the only control method beingused by local growers and it has caused environmentalconcerns leading to find non-chemical control methods suchas biofumigation.It has been shown in numerous studies that the use ofBrassica crops as green manure provides promising alter-natives to synthetic chemical fumigants (Brown and Morra,1997). Brassica crops are considered as break crops becausethey are able to break the life cycle of several soil bornepathogens such as wheat take-all (Brennan and Murray,1988). They have been proved to be effective againstnumerous nematodes (Buskov et al., 2002), fungal plantpathogens (Kirkegaard et al., 1996) and weeds (Boydston andHang, 1995). These biocontrolling effects are attributed to theproduction of volatile biotoxic compounds through a processwhich is known as biofumigation. The cruciferous tissuespossess thioglucoside compounds known as glucosinolates(GSLs), the secondary polar sulfur metabolites, which are thereason for the sharp or biting taste of condiments or theflavors of different edible Brassica tissues (McGregor et al.,1983). The GSLs are not harmful to organisms by themselvesbut they can change into isothiocyanates (ITCs), nitriles,thiocyanates, and oxazolidinethiones and other toxic com-pounds (Bending and Lincoln, 1999) by the endogenousenzyme myrosinase when Brassica tissues are damaged(Bones and Rossiter, 1996). The quality of the hydrolysisproducts depends on the type of organic side chain on theparent molecule and the environmental conditions (Rosa etal., 1997). No research has been conducted on the presence andidentification of GSLs in Iranian cultivars of Brassicas. Theobjective of this study was to evaluate the potential of bio-fumigation using three local Brassica crops against a numberof S. sclerotiorum isolates by in vitro experiments.Firstly, the inhibitory effects of Brassica tissues on radialgrowth and sclerotia formation of S. sclerotiorum weredetermined. Five highly aggressive isolates of S. sclerotiorum(Sc18d; Bahar, Sd6b; Bahar, Sd21n; Lalehjin, Sc59k; Baharand Sa21b; Lalehjin) were obtained from the CultureCollection of Plant Pathology Laboratory, Agriculture College,BuAli Sina University, Hamadan, Iran. These isolates hadalready been sampled in five heavily infected potato fields inHamadan province. The isolates were routinely cultured onpotato dextrose agar (PDA, 39 g/L; Merck, Darmstadt,Germany) and stored at 4 ± 1

Sixteen isolates belonging to 11 species of Trichoderma (T. asperellum, T. ceramicum, T. andinensis, T. orientalis, T. atroviride, T. viridescens, T. brevicompactum, T. harzianum, T. virens, T. koningii and T. koningiopsis) were evaluated for biological control of potato (Solanum tuberosum) stem rot caused by Sclerotinia sclerotiorum. In dual culture tests, all antagonists significantly reduced sclerotia formation, and were able to inhibit radial growth of the pathogen. Growth inhibition by production of volatile and non-volatile inhibitors was also measured in in vitro tests. In screening the most efficient species of Trichoderma, establishment of mycelium on sclerotia and sclerotia lysis were also considered as important biocontrol qualities. Excluding T. asperellum, T. brevicompactum, T. andinensis and T. harzianum, all tested Trichoderma species were able to lyse sclerotia. The sclerotia-destroying species of Trichoderma and one isolate of Talaromyces flavus were tested in greenhouse tests and during 2 years of field experimentation during the 2007 and 2008 cropping seasons. After one aerial application of spore suspension in greenhouse trials, T. koningii, T. virens, T. ceramicum and T. viridescens were the most effective bio-agents and reduced significantly disease severity, and the least biocontrol efficacy was observed in T. flavus. Under field conditions and after five soil and foliar applications of spore suspension, all tested antagonists reduced significantly disease incidence. T. viridescens followed by T. ceramicum showed the best results. T. flavus and T. orientalis were less effective than other tested antagonists in both field trials.

This study was conducted in Bahar and Lalehjin, Hamadan, Iran to assess the biocontrol efficacy of Coniothyrium minitans Campbell against potato white mold caused by Sclerotinia sclerotiorum (Lib.) de Bary under field and greenhouse conditions. In addition, the resistance of common potato cultivars against S. sclerotiorum was determined in a greenhouse experiment. After straw inoculation of six potato cultivars (Pashandi, Istambouli, Agria, Marfauna, Alpha and Spartaan) with S. sclerotiorum, the least disease severity was observed in Spartaan and Marfauna. Agria showed the most susceptibility to S. sclerotiorum. Compared with the healthy control, different concentrations of C. minitans conidia (10 , 10 and 10 conidia/mL) reduced disease severity under greenhouse condition, and a concentration 10 was the most effective treatment. During 2008 and 2009, four field trials were conducted to evaluate the efficacy of C. minitans in different soil and aerial applications on disease incidence of potato white mold. In 2008, soil application of Contans WG (a commercial product of C. minitans) showed the greatest biocontrol capacity whereas soil application of solid-substrate C. minitans was found inferior when compared with other treatments in both Bahar and Lalehjin field sites. In 2009, benomyl application was the most effective treatment in reducing disease incidence in both tested field sites.

Aims The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae. Methods and Results Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples. Conclusions This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens. Significance and Impact of the Study This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies.

This study was conducted to evaluate the potential of biofumigation in three Brassica crops including Brassica napus, Brassica juncea and Brassica campestris against potato stem rot caused by Sclerotinia sclerotiorum in field tests. Results from field trials carried out in three naturally infected potato fields during three cropping seasons of 2008–2010 showed that the Brassica crops used as green manure cover crops were able to significantly reduce disease incidence and mean percentage of dead plants (as a proportion of infected plants). Although results varied somewhat by field site and year, B. juncea generally provided the highest level of control, averaging greater than 55.6 % reduction in disease incidence over all fields and years, compared to average disease reductions of 31.6 and 45.8 % for the B. napus and B. campestris crop treatments, respectively. Furthermore reduction of dead plants averaged 61.6, 39.2 and 32.1 % for B. juncea, B. napus, and B. campestris, respectively. In this study, Brassica crops showed various significant inhibitory effects in different fields and years indicating that disease development is affected by other factors including environmental conditions.

Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town, Jinyun County, Zhejiang Province of China. In this paper, ultrastructural assessments on fungus-host interactions and teliospore development are presented, and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon. Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction, and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane. Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium. In addition, internal transcribed spacer (ITS) and large subunit (LSU) sequence data showed that U. coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa (Poaceae).

This study was conducted to assess the biocontrol efficacy of Bacillus subtilis (strain 4812) and Bacillus methylotrophicus (strain H8) individually or in combination with two plant defense inducers including Acetyl salicylic acid (ASA) and DL-Beta-aminobutyric acid (BABA) against tomato wilt caused by Ralstonia solanacearum. The pathogen was significantly inhibited by all treatments in the greenhouse and in vitro tests. The treatments H8+ASA and 4812+H8 were found to be the most effective treatments in in vitro tests. Applied on tomato seeds and as soil drenching, the disease was most inhibited by H8 whereas 4812+H8+ASA was the least effective treatment. High activity of phenylalanine ammonia-lyase was observed in the plants treated with 4812+H8, H8+ASA and 4812+H8+ASA. The highest expression of peroxidase and polyphenoloxidase enzymes was found in the plants treated with H8, 4812+H8 and H8+ASA. The result of real time PCR showed that concentration of the pathogen in stem tissues was significantly reduced in all treated plants and H8+ASA was the most effective treatment. This study revealed that combined application of Bacillus strains can be considered as a more effective biocontrol agent against tomato bacterial wilt. Furthermore, the synergistic effect of plant defense inducers was variable to different Bacillus strains.