Biological control of Dutch elm disease: Larvicidal activity of Trichoderma harzianum, T. polysporum and Scytalidium lignicola in Scolytus scolytus and S. multistriatus reared in artificial culture

A method is described for the culture of larvae of Scolytus multistriatus and Scolytus scolytus on an artificial medium following exposure to cultures of microorganisms. In control cultures, a natural mortality rate of 21.2% was found for S. multistriatus and 17.6% for S. scolytus. The effects of Trichoderma harzianum, T. polysporum and Scytalidium lignicola on fifth instar larvae of S. scolytus and S. multistriatus reared on the artificial medium were studied. The fungi were larvicidal and larval mortality was increased to more than 80% by inoculation of the larvae with the fungi. Another fungus, Phomopsis oblonga, had little effect on larvae of S. scolytus. The results are discussed in relation to mechanism of pathogenicity of the fungi and their potential use in the control of Dutch elm disease. It is proposed that with modifications, the method is applicable to other bark beetle pests.     

Effect of nutrition of parasites with nectar of melliferous plants on parasitism of the elm bark beetles (Col., Scolytidae)

In the forest of Kljestevica, the elm is attacked by four species of elm bark beetles: Scolytus multistriatus (Marsh.), Scolytus scolytus (Fab.), Scolytus pygmaeus (Fab.) and Scolytus ensifer (Rich.) (Col., Scolytidae). Scolytus multistriatus and S. pygmaeus are the most numerous species. Elm bark beetle is parasitized by four species of parasites: Dendrosoter protuberans (Nees), Ecphylus silesiacus (Ratz.), Coeloides scolyticida (Wesm.) (Hym., Braconidae) and Entedon leucogramma (Ratz.) (Hym., Eulophidae). Ecphylus silesiacus and D. protuberans are the most numerous species. The nectar of mustard ( Sinapis alba L., Brassicaceae) flowers, sweet basil ( Stachys recta L., Lamiaceae) flowers and of wild carrot ( Daucus carota L., Umbelliferae), has an important effect on the number of parasites and on the parasitism of the elm bark beetles. Wild carrot is the most attractive plant to the parasites for additional nutrition, especially for D. protuberans . Mustard and sweet basil flowers are suitable for additional nutrition of E. silesiacus imagos. On meadow flowers growing spontaneously in forest clearings, the lowest number of parasite imagos was collected by an entomological catcher, and the lowest percentage of parasitized larvae and eggs of the elm bark beetle was recorded in elm control catch trunks that were not near the cultivated melliferous plants.     

Isolation of Bacillus thuringiensis subsp. israelensis from diseased field-collected larvae of the saturniid moth, Hylesia metabus, in French Guiana

Abstract An isolate of Bacillus thuringiensis subsp. israelensis (BTI) was obtained from field-collected larvae of the saturniid moth, Hylesia metabus . This isolate was shown to belong to serotype H 14, and to produce a spherical parasporal body identical to that of the type strain of BTI. With an LC₅₀ of 0.764 μg cm⁻², this isolate was more toxic to H. metabus than the HD-1 strain of B. thuringiensis subsp. kurstaki (HD-1). These results demonstrate that BTI can be active against lepidopterous insects, a wider spectrum of activity than previously realized.     

Control of Plutella xylostella using polymer-formulated Steinernema carpocapsae and Bacillus thuringiensis in cabbage fields

Abstract:  Field trials evaluating the potential of the entomopathogenic nematode Steinernema carpocapsae and the feasibility to combine nematodes with Bacillus thuringiensis for sustainable control of the diamondback moth (DBM) Plutella xylostella were conducted in cabbage cultivated in the province Probolinggo, east Java and Indonesia. A single use of 0.5 million S. carpocapsae  m⁻² applied with a surfactant-polymer-formulation containing 0.3% xanthan and 0.3% Rimulgan^® achieved a significant reduction of the insects per plant with >50% control after 7 days. Even 14 days after the application about 45% control was recorded and dead larvae containing nematodes were found. No significant effects were recorded when the formulation was compared with nematodes applied in water or with a surfactant alone. This was attributed to high humidity in the experimental area at the end of the rainy season and a microclimate in the cabbage heads favouring nematode survival. Weekly applications of B. thuringiensis (Turex^®) or alternating applications of Turex^® and the nematodes achieved >80% control. The application of both biological agents together every second week reached insignificant lower efficacy (70%). Nematodes can be used to substitute ineffective chemical insecticides and alterations with B. thuringiensis can prevent the further development of resistance against the bacterial control agent.     

Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

Fusarium solani, a weak pathogen of the larval stages of the large elm bark beetle Scolytus scolytus (Coleoptera: Scolytidae)

Fusarium solani, a common colonist of beetle-infested elm bark, has been isolated from dead larvae and adults of the large elm bark beetle, Scolytus scolytus. Two infectivity tests indicated that this fungus is probably a weak pathogen of the larval stages of the beetle. Total mortality was not achieved in either test, but the mortality associated with fungal inoculation was occasionally significantly greater than the mortality in the uninoculated controls in both experiments (P<0.05, experiment A; P<0.025, experiment B). Disease symptoms suggest that the larvae attacked by F. solani may have died from bacterial infection.     

Biology of Campoletis chlorideae (Uchida) (Hym., Ichneumonidae) developing in Bt-treated, Bt-resistant Helicoverpa armigera (Hübner) (Lep., Noctuidae) larvae

Abstract:  Life history parameters in two generations of endoparasitoid Campoletis chlorideae (Uchida) were examined using Bacillus thuringiensis (Bt)-resistant Helicoverpa armigera (Hübner) larvae feeding on B. thuringiensis toxin Cry1Ac. In the laboratory, Bt toxin was fed to Bt-resistant host larvae continuously in case of Bt treatment and only before or after the host larvae were parasitized in Bt–P and P–Bt treatments, respectively. C. chlorideae pupae developed faster in Bt treatment than non-Bt treatment. The shortened pupal stage duration was mainly because of the feeding of host larvae on Bt-diet before being parasitized. Body length of adult male C. chlorideae developed inside Bt-treated Bt-resistant (Bt–Bt) H. armigera larvae significantly decreased, especially in host larvae feeding on Bt-diet after being parasitized. However, survival, pupal mortality and adult longevity of C. chlorideae were almost unaffected in Bt-resistant H. armigera larvae feeding on Bt-toxin. Furthermore, Bt-treated host larvae had the same effect on the F₁ progeny of C. chlorideae as the previous generation, and there was no significant difference between generations. This experiment suggests that there is very limited effect on the life history parameters in two generations of C. chlorideae parasitizing Bt–Bt H. armigera larvae. But both generations of C. chlorideae are affected when Bt-resistant H. armigera larvae fed on Bt toxin for different durations.     

Isolation and phenotypical identification of spore-forming Bacillus species from Jordanian habitats with larvicidal activity against Drosophila melanogaster

Spore-forming Bacillus isolates were recovered from different Jordanian habitats. Of 37 samples, 187 colonies were selected. Forty-six (24·6%) of them were pathogenic to the third instar larvae of Drosophila melanogaster . Larvicidal activity of the isolates was from 0% (non-cultivated soil) to 83·3% (decomposed animal residues). The total spore count per gram weight varied from 0·1 × 10⁵–18 × 10⁵ among the 37 tested samples. Morphological and microscopical identification of the isolates showed the presence of 16 different Bacillus species. The pathogenic isolates were B. thuringiensis (44) and B. sphaericus (2).     

Effect of biological and chemical insecticides on Spodoptera species (Lep., Noctuidae) and marketable yields of tomatoes

Various biological and chemical insecticide treatments were evaluated against beet armyworm Spodoptera exigua (Hübner), yellowstriped armyworm Spodoptera ornithogalli (Guenée) and southern armyworm Spodoptera eridania (Cramer) to determine their effects on Spodoptera species, fruit quality and marketable yields of tomatoes Lycopersicon esculentum Mill. Biological insecticides included several Bacillus thuringiensis (Berliner) products including Condor^® OF, Dipel^® 2X, Javelin^® WG, Bactec^® III, Biobit^® FC, Cutlass^® WP and Lepinox^® G. Other biological treatments included a baculovirus, SeNPV isolated from S. exigma , and an entomophagous nematode, Steinernema carpocapsae Weiser. Chemical treatments consisted of several insecticides from various classes including a carbamate (methomyl), pyrethroid (fenpropathrin) and an organophosphate (chlorpyrifos). A natural extract from the neem tree azadirachtin and an untreated control were also included in the evaluation studies. Population densities of S. exigua were below the economic threshold level in control plots and there were no significant differences for small, medium and large larvae. Densities of S. ornithogalli and S. eridania larvae exceeded the threshold level and significant treatment differences were observed in their populations. Fruit injuries were significantly higher in nonefficacious treatments that included S. carpocapsae , SeNPVs and untreated controls. Average weight per fruit was not significantly affected by treatment rates of applications, but total marketable yields were significantly higher in efficacious biological, chemical and combination treatments compared with the control.     

Respective role of the 42- and 51-kDa components of the Bacillus sphaericus toxin overexpressed in Bacillus thuringiensis

Abstract The 42- and 51-kDa protein genes of Bacillus sphaericus 1593 have been subcloned independently downstream from the cytA gene promoter of Bacillus thuringiensis serovar israelensis and introduced into a non-mosquitocidal strain of Bacillus thuringiensis . Consequently, each protein was overproduced and accumulated as inclusion bodies which were purified. For the first time, the 42-kDa protein inclusions alone were found to be toxic to Culex pipiens larvae (LC₅₀ at 48 h 300 ng ml⁻¹); in contrast, the 51-kDa protein inclusions were not. Moreover, a synergistic effect between these two components was observed.     

Characterization and toxicity of Bacillus thuringiensis isolates from warehouses to Spodoptera exigua (Lep., Noctuidae)

Bacillus thuringiensis was isolated from 122 of 413 samples obtained from warehouses. Eighty-seven (71.31%) of these B. thuringiensis isolates were toxic to Spodoptera exigua , causing more than 60% mortality. Twenty-seven isolates were highly toxic to S. exigua , causing more than 95% mortality. Isolates 133, 47 and 58, which belonged to serotype H₇, H₄, H₄, respectively, were more active than the other isolates and their 50% lethality concentration (LC₅₀) values were 17.93, 14.78 and 15.55  μ g/ml, respectively. The isolate 133, 47 and 58 were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and polymerase chain reaction. The results showed that they contained ~135 and 65 kDa crystal proteins, that were similar with reference strain HD-1. Isolate 133 contained the cryIA(a) , cryIA(b) , cryIA(c) , cryIE and cryII genes whereas both isolate 47 and 58 contained the cryIA(b) , cryIA(c) , cryIE and cryII genes; but they did not contain cryIII gene.     

Relative toxicity of native Chilean Bacillus thuringiensis strains against Scrobipalpuloides absoluta (Lepidoptera: Gelechiidae)

The larva of Scrobipalpuloides absoluta , a South American moth, is the most devastating insect pest of tomato production in Chile. The potential for using bacterial insecticides was studied analysing the relative toxicity of native Bacillus thuringiensis (BT) isolates belonging to the Chilean collection. The polymerase chain reaction (PCR) technique was used in order to facilitate the prescreening. Mixtures of homologous specific primers to regions within genes encoding CryI , CryIII and CryIV crystal proteins were employed to generate a PCR product profile of each BT isolate. Four isolates were selected and further characterized by means of SDS-PAGE, Western blot and bioassays on fourth-instar S. absoluta larvae. Relative toxicities were evaluated by LD₅₀ determinations. The entomocidal activity of isolate 121e, an autoagglutinating strain, was threefold higher than toxin synthesized by B. thuringiensis var. kurstaki . This native strain was also active against Culex pipiens larvae, although much less than towards S. absoluta .     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.     

Laboratory screening of different Bacillus thuringiensis strains against certain lepidopteran pests and subsequent field evaluation on the pod boring pest complex of pigeonpea (Cajanus cajan)

Laboratory evaluation of different Bacillus thuringiensis subspecies revealed that B. thuringiensis subsp. kurstaki (NCIM 2514) at 10⁸ spores/ml concentration caused more than 85% mortality to the neonate larvae of the lepidopteran insects Spodoptera litura (F.) and Phthorimaea operculella (Z.). This strain at 10¹⁰ and 10⁸ spores/ml concentration was effective against the major lepidopteran pests comprising the pod boring complex of pigeonpea (Cajanus cajan), viz. Helicoverpa armigera (H.) and Exelastia atomosa (W.) under the field trials. Total grain yield from this treatment was at least 1.5 times more than the untreated control.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Toxicity of Bacillus thuringiensis and protoplast fusant against Spodoptera litura (F.)

U.S. PUNTAMBEKAR, S.N. MUKHERJEE AND P.K. RANJEKAR. 1995. The octopine-positive tumorogenic intergeneric hybrid (AB0242) derived by protoplast fusion between Agrobacterium tumefaciens and Bacillus thuringiensis was found to be toxic towards the lepidopteran larvae of Spodoptera litura . Twenty-four-hour-grown cells of AB0242 showed 80% mortality, whereas a spore suspension of the parent B. thuringiensis , grown for 72 h, exhibited 93% mortality when tested against the neonate larvae of the test insect.     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity

Abstract The mosquitocidal parasporal bodies of the PG-14 isolate of Bacillus thuringiensis ssp. morrisoni and B. thuringiensis ssp. israelensis were purified on sodium bromide gradients and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electron microscopy and bioassays against mosquito larvae. The parasporal bodies of both subspecies were spherical/ovoidal, approx. 0.7–1.2 μm in diameter, and contained major proteins of 28, 65, 126 and 135 kDa. In addition to these, the parasporal body of B. thuringiensis ssp. morrisoni contained at least one other major protein, of 144 kDa, which correlated with the presence of a quasi-bi-pyramidal inclusion not present in the B. thuringiensis ssp. israelensis parasporal body. The LC₅₀ for parasporal bodies of each subspecies was in the range of 3 ng/ml for fourth-instars of Aedes aegypti . These results indicate that B. thuringiensis Serotype 8a:8b, which is generally considered to produce proteins toxic to lepidopterous insects, is capable of producing a protein toxin complement similar to B. thuringiensis Serotype 14.