Isolation of pathogenic bacteria from <Emphasis Type="Italic">Oberea linearis</Emphasis> (Coleptera: Cerambycidae)

The bacterial flora of the Oberea linearis (Coleoptera: Cerambycidae) was investigated and 13 different bacteria were isolated from O. linearis larvae. Seven of these bacteria were performed and characterized at species level and the rest of them were characterized at genus level. In this study, we determined morphological and physiological characteristics of the bacterial isolates by conventional and routine techniques, biochemical properties and metabolic enzyme profiles by API20E and Phoenix 1000A panel test systems. Additionally, 16S rRNA gene sequence analysis was also performed to identify the isolates at the molecular level. The isolates were identified as Acinetobacter calcoaceticus (Ol1), Enterobacter aerogenes (Ol2), Pseudomonas sp. (Ol3), Flavobacterium sp. (Ol4), Microbacterium sp. (Ol5), Enterobacter agglomerans (Ol6), Xanthomonas sp. (Ol7), Pseudomonas syringae (Ol8), Pseudomonas sp. (Ol9), Xanthomonas sp. (Ol10), Enterobacter cancerogenus (Ol11), Xanthomonas maltophilia (Ol12), and Serratia marcescens (Ol13). This is the first record of bacterial isolates (Ol5, Ol8, Ol11, Ol12) from any insect. All these bacteria were tested against O. linearis larvae, and Serratia marcescens was found to cause the highest mortality (65%). On the other hand, we determined 90% mortality against this pest within four days by utilizing spore and crystal mixture of Bacillus thuringiensis isolated from Melolontha melolontha.     

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle,Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae)

Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×10⁶ conidia mL^–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.     

Determination of the bacterial flora as a microbial control agent of <Emphasis Type="Italic">Toxoptera aurantii</Emphasis> (Homoptera: Aphididae)

Toxoptera aurantii (Homoptera: Aphididae) is one of the most important pests of many agricultural plants such as camellia, cocoa and coffee worldwide. The culturable bacterial flora of the pest was determined to find new microbes that can be used as biocontrol agent against T. aurantii. A total of six bacteria were isolated and identified by a variety of morphological, physiological, biochemical and molecular tests. In addition, an approximately 1,150 bp fragment of the 16S rRNA gene region was sequenced to verify isolate identification. According to the identification studies, the isolates were identified as Bacillus tequilensis Cb1, Chryseobacterium stagni Cb2, Pseudomonas flourescens Cb3, Rahnella aquatilis Cb4, Staphylococcus sp. Cb5 and Cb6. Pathogenicity of the bacterial isolates were carried out against the last instar nymphs of T. aurantii. The highest activity was obtained from Pseudomonas flourescens Cb3 with 50% mortality within 10 days after application (p<0.05). Mortalities of other treatments ranged from 6.6 to 20%. The results presented here show that Pseudomonas flourescens Cb3 appears to be a significant candidate as a possible biocontrol agent against T. aurantii and should be beneficial in the future biocontrol programs of the pest.     

Isolation and characterization of a Serratia marcescens with insecticidal activity from Polyphylla olivieri (Col.: Scarabaeidae)

Members of the genus Serratia are known for their abilities to infect insects. In this study, a red‐pigmented S. marcescens was isolated and characterized from the infected larvae of Polyphylla olivieri using bacterial cultivation, phylogenetic analysis as well as bioassays against larvae of the two insect pests, Plodia interpunctella and Ephestia kuehniella. Comparative 16S rRNA and groEL gene sequence BLAST analyses strongly suggested that the isolated strain should be placed in the genus Serratia, sharing high sequence similarities with several strain of S. marcescens associated with insects. Phylogenetic analysis placed the isolated bacterium with other S. marcescens bacteria in a clade with high bootstrapping values. To assess pathogenicity of the S. marcescens isolate, the bacterial cells were either injected into the haemolymph of the fifth‐instar larvae or added to the diets of insects. Survival curves of the control insects and those challenged with six different concentrations of S. marcescens showed that the S. marcescens isolate significantly reduced survival rates of the larvae. The LC₅₀s of the bacterium on P. interpunctella and E. kuehniella were 1992.26 and 1.09 × 10⁴ (CFU/μl) for injection bioassays at 6 h post‐injection, and 4.48 × 10⁴ and 1.96 × 10⁵ (CFU/10 μl) for feeding bioassays at 24 h post‐feeding, respectively. Injection of the bacterial culture supernatant into the larvae led to continuous bleeding from the site of injection, while injection of heat‐treated culture supernatant of the bacterium did not cause continuous bleeding. Together, our results showed the possibility of using this S. marcescens isolate in microbial control of the insect pests after addressing the safety concerns. Moreover, it might be considered as a source of useful bioactive molecules and genes with application in insect control and biotechnology via developing insect‐resistant plants.     

Screening of entomopathogenic fungi against the European spruce bark beetle,Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera: Scolytidae), is one of the most serious destructive pests of oriental spruce (Picea orientalis L.). In order to find an effective biocontrol agent against this pest, we determined the effectiveness of entomopathogenic fungi against D. micans. Virulence of nine highly pathogenic strains including Beauveria bassiana (2), Beauveria cf. bassiana (2), Metarhizium anisopliae (2), Metarhizium sp. (1), Isaria fumosorosea (1) and Evlachovaea sp. (1) was evaluated on D. micans larvae and adults under laboratory conditions. Mortality values for both larvae and adults ranged from 83 to 100% and from 23 to 100%, respectively. Larvae were more susceptible to fungi than adults (P<0.05). Based on screening tests, B. cf. bassiana isolate KTU-53 was found the most effective isolate. LC₅₀ values were calculated as 1.77×10⁴ and 2.65×10⁴ conidia mL^?1 for isolate KTU-53 against larvae and adults, respectively. Consequently, B. cf. bassiana isolate KTU-53 appears to be the most promising microbial control agent for biocontrol of D. micans.     

Isolation screening and characterisation of local beneficial rhizobacteria based upon their ability to suppress the growth of Fusarium oxysporum f. sp. radicis-lycopersici and tomato foot and root rot

Local beneficial rhizobacteria were selected based upon their ability to control the fungus Fusarium oxysporum f. sp. radicis-lycopersici which causes crown and root rot of tomato. Seven out of 384 strains prevailed in multiple and dual cultures and were identified as Pseudomonas chlororaphis (one strain), Bacillus cereus (one strain), Serratia marcescens (three strains) and Serratia rubidaea (two strains), by sequencing the 16S rRNA or the 16S and 23S rRNA inter-spacer region. The seven selected rhizobacteria were tested for their biocontrol and growth-promoting effects in planta, and their antifungal properties in vitro. All strains significantly reduced disease severity under controlled conditions, in a gnotobiotic system and in pots. Moreover, one P. chlororaphis and one S. marcescens strain significantly decreased disease severity to the level of the healthy control under natural conditions in pots experiments. The inhibitory activity of bacterial liquid cultures' metabolites on the fungus was demonstrated for all strains in vitro, using filter paper, thin layer chromatography and microtiter bioassays. Genes encoding phenazines were tentatively detected by PCR in the P. chlororaphis strain and chitinase-encoding genes were detected in one S. rubidaea and all three S. marcescens strains. Production of phenazine-1-carboxamide and hydrogen cyanide was evidenced for the P. chlororaphis strain while protease activity and production of siderophore-like compounds was confirmed in all bacterial strains. Possible use of these strains as biological control agents and their impact on natural biocontrol of pathogens in soils is discussed.     

Identification of a bacterium isolated from the diseased brown planthopper and determination of its insecticidal activity

The brown planthopper, Nilaparvata lugens, is one of the most harmful insect pests of rice crops in Asian countries. To find an effective biological control agent against this pest, we investigated the bacterial flora of field N. lugens collected from Jiangsu Province, China, in 2012 and tested its insecticidal activity. A novel bacterium strain, S-JS1, was isolated from N. lugens nymphs and adults and showed a high level of insecticidal activity. Based on its phenotypic, physiological and biochemical properties, and its 16S rRNA gene phylogeny, the isolate was assigned to Serratia marcescens; the name S. marcescens S-JS1 is proposed. The pathogenicity of S-JS1 against the third-instar nymphs, and the macropterous and the brachypterous adults of N. lugens were compared. The median lethal concentration (LC₅₀) values of S-JS1 against the brachypterous adult were the lowest (LC₅₀, 1.53?×?10⁸ colony forming units (cfu)/ml), followed against the macropterous adult (LC₅₀, 1.65?×?10⁹?cfu/ml) and third-instar nymphs (LC₅₀, 1.86?×?10⁹?cfu/ml) at 5 days post-infection. The median lethal time values of 8?×?10⁸?cfu/ml S. marcescens S-JS1 against the brachypterous adult, macropterous adult, and third-instar nymph were 4.5, 5.5, and 5.7 days, respectively. These results indicate that the S-JS1 isolate appears to be a promising S. marcescens strain with strong biocontrol potential against N. lugens.     

Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.     

Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Several pathogenic bacteria strains were isolated from dead larvae of the weevil, Rhynchophorus ferrugineus Oliver, collected on palm trees in Taiwan, China. Strains of pure cultures were temporarily named TW‐1, TW‐2, TW‐3, TW‐4, TW‐5, TW‐6, TW‐7, TW‐8, TW‐9, TW‐10, TW‐11, TW‐12, TW‐13 and TW‐14. These strains were identified by synthetically using colony morphological characters, physiological and biochemical tests and molecular biological analysis. Our results showed that these 14 isolated strains belonged to four genera and five species. The expected approximate 1400‐bp bands were obtained through 16S rDNA universal primer amplification. The sequencing results obtained from a commercial sequencing company were initially compared with a corresponding database from NCBI using BLAST, and a phylogenetic tree was constructed using mega 5.1 software. The phylogenetic results indicated that these isolated strains had more than 99% homology with type strains. To further confirm these strains, numerous physiological and biochemical indexes were confirmed. TW‐1, TW‐2, TW‐3, TW‐9, TW‐12, TW‐13 and TW‐14 were identified as Serratia marcescens; TW‐4, TW‐5, TW‐8 and TW‐10 as Staphylococcus sciuri; TW‐6 as Klebsiella pneumonia subsp. pneumonia; TW‐7 as Proteus vulgaris and TW‐11 as Proteus mirabilis. The preliminary single‐dose bioassay on fourth instar larvae showed that S. marcescens had a higher virulence at a density of 1.89 OD₆₀₀ (56.37% mortality) than the other strains. The corrected mortalities of the other strains were all less than 50%. The lowest toxicities were found for P. vulgaris and P. mirabilis where the corrected mortalities were only 28.18% and 25.57%, respectively. LC₅₀ of S. marcescens was 1.2 × 10⁷ CFU/ml inoculums. Our results indicated that S. marcescens from facultative cultivable bacterial flora isolated from R. ferrugineus could potentially be used as a microbial control agent for this widespread pest.     

Isolation and identification of bacteria from <Emphasis Type="Italic">Thaumetopoea pityocampa</Emphasis> Den. and Schiff. (Lep., Thaumetopoeidae) and determination of their biocontrol potential

The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pest for pine species in Mediterranean countries including Turkey. The objective of the present study is to find a more effective and safe biological control agent against T. pityocampa. Thus, we investigated the bacterial flora of the pest insect, collected from the Middle Black Sea Region of Turkey from 2003 to 2004. Based on morphological, physiological, biochemical and molecular methods, 14 different bacterial isolates were determined. The identified bacterial flora of T. pityocampa consisted of bacteria belonging to the Enterobacteriaceae (Tp1), Arthrobacter sp. (Tp2), Staphylococcus spp. (Tp3 and 10), Bacillus subtilis (Tp4), Serratia liquefaciens (Tp5), Bacillus thuringiensis subsp. morrisoni (Tp6 and 14), an acrystalliferous form Bacillus thuringiensis (Tp7), Staphylococcus cohnii (Tp8), Bacillus licheniformis (Tp9), Bacillus pumilus (Tp11), Brevibacterium sp. (Tp12) and Bacillus simplex (Tp13). After analysing the conclusions of conventional and molecular tests Tp1 (Enterobacteriaceae), Tp2 (Arthrobacter sp.) and Tp12 (Brevibacterium sp.) were assigned as novel bacterial species. Isolate Tp5 had a promising insecticidal effect on third instar larvae of T. pityocampa (up to 70% mortality within 10 days).     

Entomopathogenicity of endophytic <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain SRM against larvae of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Noctuidae: Lepidoptera)

An endophytic Serratia marcescens strain SRM (MTCC 8708) isolated from the flowers of summer squash was found to be entomopathogenic against the larvae of Helicoverpa armigera. Natural epizootic of this bacterial strain on the larvae collected from summer squash flowers ranged from 19.9 to 72.3%. Under laboratory conditions, a dose of 6 × 10¹⁰ c.f.u./ml diet induced 66.3% mortality of first instar H. armigera larvae. Similarly all other growth and development parameters of the insect were severely retarded in a dose-dependent manner. The bacterium invaded the entire alimentary canal and haemolymph with successful replacement of all other gut-associated microflora. There was a great reduction in midgut proteinase activity due to inhibition of five major proteinase isozymes by S. marcescens infection. Further, a synergistic interaction between chitinases isolated from this strain and Bacillus thuringiensis Cry1Ac toxin was observed. The present findings suggest that this plant-associated S. marcescens strain SRM could be suitably exploited for the management of H. armigera.     

A modified semi-synthetic diet for bioassay of non-sporeforming entomopathogenic bacteria against Spodoptera litura

Standard semi-synthetic diet used for rearing Spodoptera litura (Fab) larvae contains many antimicrobials. This diet is not suitable for evaluating non-sporeforming bacteria. We determined the effect of antimicrobials present in S. litura semi-synthetic diet on growth of entomopathogenic Serratia marcescens. Nutrient broth supplemented with four and two times reduced doses of sorbic acid (0.15 g) and methyl parahydroxybenzoate (0.05 g) supported bacterial growth comparable to control medium. No growth could be observed even at five times reduced concentrations of formaldehyde and streptomycin sulphate. Diet for rearing S. litura was modified accordingly and validated using two S. marcescens strains. Larval mortality ranged between 20% and 30% on the standard diet and more than 90% in the modified diet.     

Isolation,characterization and virulence of bacteria from <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.     

Biological control potential of native entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) against Spodoptera cilium (Lepidoptera: Noctuidae) in turfgrass

In laboratory studies, we demonstrated that five native entomopathogenic nematode species/isolates caused 100% mortality of Spodoptera cilium larvae, a soil surface-feeding pest of turfgrass. At 25 infective juveniles/cm² applied to sod, two selected Turkish species, Steinernema carpocapsae and Heterorhabditis bacteriophora (Sarigerme isolate), averaged 77% and 29% larval mortality, respectively.     

Isolation and virulence of entomopathogenic fungi against larvae of hazelnut weevil Curculio nucum (Coleoptera,Curculionidae) and the effects of combining Metarhizium anisopliae with entomopathogenic nematodes in the laboratory

An approach to ensure effective pest biocontrol would be to select native isolates of biological control agents (BCAs). A survey to isolate entomopathogenic fungi (EPF) from a hazelnut growing area has been carried out. EPF were recovered from 133 of the 295 soil samples. The main species isolated were Metarhizium anisopliae sensu lato (36%) and Beauveria bassiana sensu lato (42.8%). With regard to controlling abiotic factors, altitude had an effect on the distribution of B. bassiana, but not on M. anisopliae. Cropping system did not have an effect on the occurrence of these EPF, while pH appeared as a predictive variable for both. In addition, we tested the virulence of six of these isolates: three M. anisopliae and three of B. bassiana against larvae of Curculio nucum L. The highest larval mortality (reaching 80%) was due to M. anisopliae (strain 34) when applied in simultaneous combination with four entomopathogenic nematode species: Steinernema carpocapsae (strain B14), Steinernema feltiae (strain D114), Steinernema. sp. (strain D122) and Heterorhabditis bacteriophora (strain DG46). The effect of nematodes was greater and no antagonistic or synergistic effects were observed.     

Biocontrol efficacy of protoplast fusants between Bacillus thuringiensis and Bacillus subtilis against Spodoptera litura Fabr.

Bacillus thuringiensis (Bt) is a microbial pesticide widely used to control crop pests. Its strains have good biocontrol activity against crop insect pest, but lack some desirable characteristics that are found in Bacillus subtilis. An attempt has been made to combine those desirable characteristics; we used a highly effective biocontrol strain of B. thuringiensis in protoplast fusions with a strain of B. subtilis. The fusants were identified through cell culture and stained with crystal violet. The Bt and B. subtilis protoplasts were induced to fuse by PEG 6000. The fusants were produced almost 95% mortality in first instar larvae of Spodoptera litura. The lethal doses (The LC₅₀ and LC₉₀) for mortality of S. litura values were significantly in lower level in the fusant-treated larvae, when compared with Bt and B. subtilis individual treatment. The consumption and digestion of S. litura significantly decreased after treatment with fusant. Also the approximate digestibility of S. litura increased significantly.     

Field evaluation of eco-friendly biocontrol package for the management of pod borer,Helicoverpa armigera Hubner,and fusarium wilt in chickpea,Cicer arietinum

Helicoverpa armigera is a serious pest of chickpea and causes great damage to crop. Extensive and indiscriminate use of insecticides has led to the development of resistance in H. armigera. Among the several alternative methods for management of H. armigera, the nuclear polyhedrosis virus (NPV) is promising, whereas Trichoderma sp. have shown promising results against chickpea wilt. The experiments to evaluate biocontrol package against H. armigera and wilt disease under field conditions were conducted. Lowest H. armigera larval population (0.71 larvae/plant) was recorded in chemical control, which was at a par with biocontrol package (0.91 larvae/plant), and both the treatments were significantly better than control. Lowest per cent pod damage (3.85%) was recorded in chemical control followed by biocontrol treatment (5.08%) and unsprayed control (8.61%). The yields from biocontrol package (13.45 q/ha) and chemical control (15.37 q/ha) were significantly higher than unsprayed control (10.7 q/ha). There was no disease incidence in all treatments in both 2008 and 2009.     

Novel strategies for the biocontrol of noctuid pests (Lepidoptera) based on improving ascovirus infectivity using Bacillus thuringiensis

Identifying novel biocontrol agents and developing new strategies are urgent goals in insect pest biocontrol. Ascoviruses are potential competent insect viruses that may be developed into bioinsecticides, but this aim is impeded by their poor oral infectivity. To improve the per os infectivity of ascovirus, Bacillus thuringiensis kurstaki (Btk) was employed as a helper to damage the midgut of lepidopteran larvae (Helicoverpa armigera, Mythimna separata, Spodoptera frugiperda, and S. litura) in formulations with Heliothis virescens ascovirus isolates (HvAV-3h and HvAV-3j). Btk and ascovirus mixtures (Btk/HvAV-3h and Btk/HvAV-3j) were fed to insect larvae (3rd instar). With the exception of S. frugiperda larvae, which exhibited low mortality after ingesting Btk, the larvae of the other tested species showed three types of response to feeding on the formulas: type I, the tested larvae (H. armigera) were killed by Btk infection so quickly that insufficient time and resources remained for ascoviral invasion; type II, both Btk and the ascovirus were depleted by their competition, such that neither was successfully released or colonized the tissue; type III, Btk was eliminated by the ascovirus, and the ascovirus achieved systemic infection in the tested larvae. The feeding of Btk/ascovirus formulas led to a great reduction in larval diet consumption and resulted in a significant decrease in the emergence rate of H. armigera, M. separata, and S. litura larvae, which suggested that the formulas exerted marked oral control effects on both the contemporary individuals and the next generation of these tested pest species.     

Pathogenicity of microorganisms isolated from the oak platypodid, <Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Murayama) (Coleoptera: Platypodidae)

The oak platypodid, Platypus quercivorus (Murayama), is the most destructive pest of Quercus and related fagaceous species in Japan. To find an effective biocontrol agent, we determined the effectiveness of entomopathogenic microorganisms against P. quercivorus. P. quercivorus-infested Oak (Quercus serrata, Q. crispula and Q. acutissima) logs were obtained from various regions in Japan, and 28 isolates of microorganisms were isolated from P. quercivorus larvae from those logs. Using molecular methods, the microbes were then classified into 18 species. After a bioassay, we found that three fungi, Lecanicillium sp., Isaria tenuipes, Beauveria bassiana, and the bacterium Serratia marcescens had the greatest potential as biological control agents against P. quercivorus. Another bioassay was then performed to determine the effect of spore or cell densities on the pathogenicity of the four microorganisms, and optimum spore or cell densities for the microbes were determined to be 10⁶ and 10⁷/ml. Based on the results and other criteria, we tentatively selected B. bassiana as the best candidate for future use against P. quercivorus larvae.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.