Lecanii Strain Research Articles

Efficacy of bio-pesticide Lecanicillium lecanii against soybean-sucking bugs Riptortus linearis during field application

Abstract. Mulyati Y, Zubaidah S, Prayogo Y. 2023. Efficacy of bio-pesticide Lecanicillium lecanii against soybean-sucking bugs Riptortus linearis during field application. Biodiversitas 24: 4829-4836. This study aimed to determine the effect of application time and surfactants on the efficacy of Lecanicillium lecanii strain Probolinggo in controlling the soybean pod-sucking bug Riptortus linearis in a field test. The study was conducted in a randomized block design consisting of two treatments, namely time of application (7 am, 10 am, 1 pm, and 4 pm) and type of surfactant (alkyl glycerol phthalate and alkylaryl polyglycol ether). Application time at 4 pm showed 93% highest mortality of R. linearis than the other treatments, with the lowest number of punctures (86 punctures) and the highest soybean dry weight (11.38 g), which were not significantly different from the application time of 10 am. The efficacy of L. lecanii increased by 74.3% after adding alkylaryl polyglycol ether. The efficacy of L. lecanii with alkyl glycerol phthalate was not significantly different from the control. In the parameters of the number of seed punctures and dry weight of soybeans, the surfactant treatment gave similar results to the control. The results showed that in the field test, the highest efficacy of L. lecanii against R. linearis occurred at 4 pm with alkil aril polyglycol ether surfactant.

Eff ect of volatile organic compounds of entomopathogenic fungi of the genus Lecanicillium and their component, the acetic acid, on the female behaviour of the western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera, Thripidae)

The e ect of VOCs of six entomopathogenic fungal strains from genus Lecanicillium on the behavioral responses of the western ower thrips females Frankliniella occidentalis on vegetative bean plants and in a Y-shaped olfactometer was studied. The signi cant repellent reactions of thrips females and a decrease in a number of o spring by 33-34% were revealed after treatment the beans by conidial suspensions of L. lecanii strains F2 and Vl 29 in the concentration of 1 × 107 spores/ml. Strains Vl 21 ( L. muscarium ) and Vit 71 ( L. attenuatum ), which showed a repellent tendency towards thrips females, also caused a signi cant decrease of o spring number. The strains isolated from aphids (ARSEF 2332 of L. dimorphum and Vl 13 of L. longisporum ) showed a tendency towards attractiveness. In the Y-shaped olfactometer the repellency of the F2 strain and the attractiveness of the ARSEF 2332 strain were con rmed. The in uence of the other studied strains on the thrips behavioral reactions was unreliable. Acetic acid, tested in the olfactometer at doses corresponding to its content in the fungal VOCs over growing mycelium, caused di erent reactions of thrips females depending on the acid concentration: attractive reactions at the dose of 0.17 ppm; neutral - at the dose of 0.34 ppm; and weak repellent - at 0.85 ppm. The obtained data indicate that acetic acid, contained in the fungal VOCs, e ect on the behavioral responses of thrips females. The repellant e ect of the fungal spores of the genus Lecanicillium on thrips females and the negative impact on o spring number increase the e ectiveness of entomopathogenic fungi.

Improvement of Akanthomyces lecanii resistance to tebuconazole through UV-C radiation and selective pressure on microbial evolution and growth arenas

Tebuconazole (TEB) is a fungicide widely used in agriculture; however, its constant application has increased the emergence of resistant plant pathogenic fungal strains and reduced the effectiveness of fungi as biological control agents; for instance, the entomopathogenic and hyperparasitic fungus Akanthomyces lecanii, suitable for simultaneous biological control of insect pest and plant pathogenic fungi, is highly sensitive to fungicides. We carried out the induction of resistance to TEB in two wild type strains of A. lecanii by UV radiation and selective pressure in increasing fungicide gradients using a modified Microbial Evolution and Growth Arena (MEGA), to produce A. lecanii strains that can be used as biological control agent in the presence of tebuconazole. Nine UV-induced and three naturally adapted A. lecanii strains were resistant to TEB at the agriculturally recommended dose, and three irradiated strains were resistant to TEB concentration ten times higher; moreover, growth, sporulation rates, production of hydrolytic enzymes, and virulence against the hemipteran Coccus viridis, a major pest of coffee crops, were not affected in the TEB-resistant strains. These A. lecanii TEB-resistant strains would have a greater opportunity to develop and to establish themselves in fields where the fungicide is present and can be used in a combined biological-chemical strategy to improve insect and plant pathogenic fungal control in agriculture. Also, the selective pressure through modified MEGA plate methodology can be used for the adaptation of entomopathogenic filamentous fungi to withstand other chemical or abiotic stresses that limits its effectiveness for pest control.

Investigation of the efficacy of some biopesticides by food exposure on Bombus terrestris L. (Hymenoptera: Apidae)

Exposure effects of Beauveria bassiana strain Bb-1 (Nostalgist), Isaria fumosorosea (= Paecilomyces fumosoroseus) strain PFs-1 (Priority), Lecanicillium lecanii strain V1-1 (Nibortem), azadirachtin (Nimbecidine) and Bacillus thuringiensis var. kurstaki (Rebound) on Bombus terrestris workers were investigated under laboratory conditions. Bombus terrestris workers were exposed to the recommended doses of biopesticides orally via feeding treated sugar syrup and treated pollen, a total of 600 individuals were used during the study. The experiments were carried out between September and November 2019 in the Research and Application laboratory of apiculture in the Department of Animal Science in Isparta University of Applied Sciences, Turkey. It was found that observed side effects differ in terms of the application methods of the biopesticides. Although worker mortalities ranged between 6 and 16% (non-toxic) in treated pollen, mortality rates 100% (highly toxic) for B.t. var. kurstaki and azadirachtin, 44% (weakly toxic) for I. f. strain PFs-1 and 8–14% (non-toxic) for other biopesticides in treated sugar syrup. The results showed that the biopesticides other than Rebound and Nimbecidine were not toxic on workers of B. terrestris in laboratory conditions.

Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica (Matsumura)

This research reported the in vitro pathogenicity of Verticillium lecanii strains, L2 and L5, and Beauveria bassiana strains, B76 and B252, against Megoura japonica using leaf-dip method. The virulence potential of these four entomopathogenic fungal strains of V. lecanii and of B. bassiana were compared between fungi conidia (concentrations 1 × 106, 1 × 107, and 1 × 108 conidia mL−1) and culture filtrate. Moreover, binary combination of four different fungal strains (L2 + B76, B76 + L5, L2 + B252, and L2 + B76 + B252 + L5) were evaluated against M. japonica under control condition. Aphid mortality was recorded after two, four, six, and eight days of post-treatment. In the conidial concentration bioassay, strain B76 showed maximal mortality (85.3%) against bean aphid, and strain L5 showed the lowest effect (60.0%) at the highest concentration (1 × 108 conidia mL−1) at eight days post-treatment. Whereas, binary combinations of B76 and L2 strains showed the highest effect against M. japonica (90.5%) than other combinations. Moreover, in comparison with the effect of filtrate and conidia bioassay, 91.4% and 84.1% were achieved in strain B76, and the lowest effect (63.8% and 55.1%) was recorded in strain L5.

Effects of Lecanicillium lecanii strain JMC-01 on the physiology, biochemistry, and mortality of Bemisia tabaci Q-biotype nymphs.

BackgroundLecanicillium lecanii is an entomopathogenic fungi, which was isolated from insects suffering from disease. Now, it is an effective bio-control resource that can control agricultural pests such as whitefly and aphids. There are many studies on the control of various agricultural pests by L. lecanii, but no report on its control of Bemisia tabaci biotype-Q exists. In this work, we studied the susceptibility of B. tabaci Q-biotype (from Ningxia, China) to L. lecanii JMC-01 in terms of nymph mortality and the changes in detoxifying protective enzymes activities.MethodsB. tabaci nymphs were exposed to L. lecanii JMC-01 conidia by immersion with the host culture. Mortality was assessed daily for all nymph stages. The detoxifying and protective enzyme activity changes, weight changes, and fat, and water contents of the nymphs were determined spectrophotometrically.ResultsAll instars of B. tabaci died after being infested with 1 × 108 conidia/mL. The 2nd-instar nymphs were the most susceptible, followed by the 3rd-instar nymphs. The corrected cumulative mortality of the 2nd- and 3rd-instar nymphs was 82.22% and 75.55%, respectively. The levels of detoxifying and protective enzymes initially increased and then decreased. The highest activities of carboxylesterase, acetylcholinesterase, peroxidase, and catalase occurred on the 3rd day, reaching 10.5, 0.32, 20, and 6.3 U/mg prot, respectively. These levels were 2.2-, 4.3-, 2.4-, and 1.4-fold the control levels, respectively. The highest activities of glutathione-S transferase and superoxide dismutase on the 2nd day were, respectively, 64 and 43.5 U/mg prot. These levels were, respectively, 2.7 and 1.1-fold that of the control level. The water and fat content in the infected B. tabaci nymphs decreased and differed significantly from the control levels. The weight increased continuously in the first 24 h, decreasing thereafter. At 72 h, the infestation level was about 0.78-fold that of the control level.ConclusionsThe studied L. lecanii JMC-01 strain is pathogenic to the B. tabaci Q-biotype. This strain interferes with the normal functioning of detoxifying and protective enzymes, and is also involved in the disruption of normal physiological metabolism in B. tabaci.

In Vitro Pathogenicity of Some Entomopathogenic Fungal Strains against Green Peach Aphid Myzus persicae (Homoptera: Aphididae)

Green peach aphid, Myzus persicae (Hemiptera: Aphididae), is an economically important pest of crops within more than 40 plant families all over the world. This study encompasses in-vitro pathogenicity of two strains of Beauveria bassiana (BB-72 and BB-252) and one strain of Lecanicillium lecanii (V-4) against green peach aphid (M. persicae). Using a leaf-dip method, three different bioassays were conducted comprised of filtrates and conidial concentrations of BB-72, BB-252 and V-4 fungal strains and their binary combinations. Infiltrate bioassays, 2 mL fungal filtrate of each strain was used. In conidial bioassays, three different concentrations (i.e., 1 × 106, 1 × 107 and 1 × 108 conidia mL−1) of each fungal strain were used, while in binary combination bioassays, LC50 and LC33 of these fungal strains were evaluated. According to the results, maximum pathogenicity against test aphids was observed at 10th day of all treatments. Both strains of B. bassiana (BB-72 and BB-252) exhibited higher mortality of aphids (up to 95 and 91%, respectively) than L. lecanii (V-4) strain (87%) in all three types of bioassays. Moreover, binary combinations of BB-72 and BB-252 strains showed significantly higher aphid mortality (94%) than other combinations. Reduced mortality in case of fungal combinations of V-4 strain might be due to the antagonistic effect of L. lecanii strain along with both strains of B. bassiana. Nevertheless, evaluation of combined pathogenicity of the fungal strains constitutes the novelty of this in-vitro study which revealed that both strains of B. bassiana (i.e., BB-72 and BB-252) are mutually compatible and can be utilized together as new biocontrol tools against destructive insect pests such as M. persicae.

High-level expression, purification and properties of an Endochitinase gene without signal peptide from Lecanicillium lecanii 43H in Pichia pastoris.

Chitinases play the key role in hydrolysis of chitin, a huge organic carbon reservoir on earth, into monomeric sugars and their eventual conversion into valuable chemicals and energy sources. The Lecanicillium lecanii strain 43H was used as the source for the Endochitinase gene without signal peptide (mchit1). This mchit1 gene was cloned and sequenced. The recombinant Endochitinase non signal peptide was overexpressed in Pichia pastoris X33 with a level of 2.048UmL-1 culture supernatant. The molecular mass of the purified recombinant Endochitinase (rmchit1) without signal peptide was 43kDa. Metal ions, detergents, and organic solvents tested indicated a significantly influence on rmchit1 activity. The obtained results demonstrated that signal peptides affect the yield expression, purification methods, recovery as well as the physicochemical properties of the enzyme.

The Herbivore-Induced Plant Volatiles Methyl Salicylate and Menthol Positively affect Growth and Pathogenicity of Entomopathogenic Fungi

Some herbivore-induced-plant volatiles (HIPVs) compounds are vital for the functioning of an ecosystem, by triggering multi-trophic interactions for natural enemies, plants and herbivores. However, the effect of these chemicals, which play a crucial role in regulating the multi-trophic interactions between plant-herbivore-entomopathogenic fungi, is still unknown. To fill this scientific gap, we therefore investigated how these chemicals influence the entomopathogenic fungi growth and efficacy. In this study, Lipaphis erysimi induced Arabidopsis thaliana HIPVs were collected using headspace system and detected with GC-MS, and then analyzed the effects of these HIPVs chemicals on Lecanicillium lecanii strain V3450. We found that the HIPVs menthol and methyl salicylate at 1 and 10 nmol·ml−1 improved many performance aspects of the fungus, such as germination, sporulation, appressorial formation as well as its pathogenicity and virulence. These findings are not only important for understanding the multi-trophic interactions in an ecosystem, but also would contribute for developing new and easier procedures for conidial mass production as well as improve the pathogenicity and virulence of entomopathogenic fungi in biological pest management strategies.

Ultrastructural exploration on the histopathological change in Phenacoccus fraxinus infected with Lecanicillium lecanii.

The histopathological changes of the second instar nymph of the mealybug Phenacoccus fraxinus infected with Lecanicillium lecanii strain 3.4505 were investigated using light, scanning and transmission electron microscopy. The results demonstrated that L. lecanii 3.4505 could infect P. fraxinus in a short period. At 24 h post-inoculation, the conidia of L. lecanii 3.4505 adhered to the indented gloves or intersegmental folds of the insect body surface. Subsequently, the germinated conidia produced germ-tubes, appressoria and extended hyphae, which tightly adhered to the cuticle. Penetration of cuticle could be achieved either by peg form appressoria or directly by hyphae. Also, the conidia and hyphae could secrete massive mucilages causing visible damage to the host cuticle. After 48 h, the body wall, tissues and organs, including cuticle, trachea, fat body, muscle, Malpighian tubules and nerve ganglion, were destroyed by ramification of hyphae as a result of infection. The endoplasmic reticulum hypertrophied and formed obvious fingerprint agglomerates, and the mitochondria swelled and deformed in the haemocytes. Finally, the mycelium fully occupied the entire haemocoel. The entire bodies were wrapped in a white mycelium, with the mycelium extending radically outward.

An extracellular antifungal chitinase from Lecanicillium lecanii: purification,properties, and application in biocontrol against plant pathogenic fungi

An extracellular antifungal chitinase from L. lecanii strain 43H was purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography; it showed a molecular mass of approximately 33 kDa with a specific activity of 167.5 U/mg protein and purification factor of 2.5. Optimum temperature and pH were observed at 40 °C and pH 6.0, respectively. This enzyme was stable at up to 40 °C and at pH 5.0-6.0. The kinetic constants K_m and V_{max} determined for the chitinase with colloidal chitin as substrate was 0.82 mg/mL and 4.51 U/mg, respectively. The presence of 5-15 mM tested metal ions led to activation of the chitinase activity with an increase of up to 126% except for Al^{3+}, Ag^+, and Hg^{2+}. Tween 80 (0.5%), Tween 20 (1%-2%), and Triton X-100 (1%) increased the enzyme activity by up to 25%, whereas higher concentration of 2% SDS completely inhibited the enzyme. The chitinase exhibited high resistance to organic solvents (methanol, acetone) and retained 89%-95% of its initial activity. The chitinase was found to inhibit the conidial germination and mycelial growth of plant pathogenic fungi. These results suggest that chitinase might be used in enzymatic reactions and as a potential fungicide against pathogens.

Pathogenicity of three entomopathogenic fungi to Matsucoccus matsumurae.

Matsucoccus matsumurae (Kuwana) (Hemiptera: Coccoidea: Matsucoccidae) is an invasive alien species and a destructive pest of two native Chinese pines, Pinus tabulaeformis Carr. and P. massoniana Lamb., throughout the eastern regions of China. The pathogenicity of three entomopathogenic fungi, Lecanicillium lecanii strain V3.4504 and V3.4505, Fusarium incarnatum-equiseti strain HEB01 and Lecanicillium fungicola strain HEB02, against M. matsumurae was tested in four instars, to evaluate their potential as a biological control agent. The results showed that the four strains caused disease and death of the scale insect, among which the L. lecanii strains V3.4504 and V3.4505 displayed stronger virulence than the F. incarnatum-equiseti strains HEB01 and L. fungicola strain HEB02 to M. matsumurae in the 2nd-instar nymphs and the adult females. Furthermore, L. lecanii V3.4505 was most virulent to M. matsumurae. The adult females and the male 3rd-instar nymphs of M. matsumurae were susceptible to L. lecanii V3.4505; the adult females were more susceptible at LT50 = 1.96 than the 3rd-instar nymphs at LT50 = 5.67. The body surface structure, cuticle thickness and wax secretions of M. matsumurae impacted the fungal infection. L. lecanii is a promising biocontrol agent, and newly emerged male 3rd-instar nymphs and adult females are a crucial period of the insect’s life cycle for M. matsumurae biocontrol.

RAPD-PCR, UP-PCR and rDNA sequence analyses of the entomopathogenic fungus Verticillium lecanii and its pathogenicity towards insects and phytopathogenic fungi

RAPD-PCR and UP-PCR analysis allowed us to differentiate Verticillium lecanii strains isolated from insect (Homoptera) and from rust fungi. UPGMA, NJ and NJ consensus trees revealed groups of isolates according to their geographical origin. Successful amplification of the ITS region occurred in a few V. lecanii strains. These ITS sequences were almost identical to known ITS sequences. Thirteen strains had similar IGS sequence of about 2.2 Kb, while strain V5 had a different and shorter (1.1 Kb) IGS-sequence. Among the isolates the correlation between conidia size and virulence towards whitefly was low. Verticillium lecanii strains were able to prevent the pustule development of Puccinia recondita. V61 showed 98% effectiveness. Strains active against wheat rust causal agent were highly virulent against whitefly and show some antibacterial activity. These strains are promising for the biocontrol of plant diseases.

Damage and Recovery from UV-B Exposure in Conidia of the Entomopathogens Verticillium lecanii and Aphanocladium album

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m−2 weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT50 (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera.

Damage and recovery from UV-B exposure in conidia of the entomopathogens Verticillium lecanii and Aphanocladium album

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m−2 weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT50 (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera.

Characterization and identification of entomopathogenic and mycoparasitic fungi using RAPD-PCR technique

Entomopathogenic and mycoparasitic fungi were characterised by RAPD technique, with special attention to evaluate the genetic stability of strains that are used as active ingredients in commercial biopesticides. Strain-specific fingerprints were constructed for Paecilomyces fumosoroseus – strain PFR 97 Apopka, Gliocladium virens – strain GL 21 and Verticillium lecanii – strain MYCOTAL. Genetic stability and homogeneity was confirmed among re-isolates that were obtained from commercial batches of bio-insecticide PFR 97TM 20%WDG and bio-fungicide SoilGardTM12G that had been produced in 1995–1999. RAPD analysis indicated the genetic identity of V. lecanii strains re-isolated from the two different bio-insecticides MYCOTAL<sup>®</sup> and VERTALEC<sup>®</sup>. The usefulness of RAPD technique was demonstrated when P. fumosoroseus strain PFR 97 Apopka was reliably identified after having passed through adults of the spruce bark beetle Ips typographus, and by analysis of the relationship between fungi of the genus Gliocladium.

Pathogenicity ofVerticillium lecanii to different developmental stages of the silverleaf whitefly,Bemisia argentifolii

Thirty-five strains ofVerticillium lecanii which originated from different hosts and geographical locations were tested as potential biocontrol agents against silverleaf whitefly,Bemisia argentifolii Bellows & Perring. All strains were tested for their pathogenicity to third-instar nymphs. Several isolates which exhibited high pathogenicity to nymphs were also tested against eggs, pupae and adults ofB. argentifolii. Eggs were found to be immune to infection, but mortality of hatching nymphs reached 95–98%. The rate of hatching nymphs’ infection depended on the age at which the eggs were inoculated and the strain’s virulence. Mortality of nymphs recorded on day 4 after inoculation varied from 0.5±0.3% to 83±2.4%; that of the control ranged from 2.5% to 10.2%. The most virulent strains, with LT50 ranging between 3.2 and 3.8 days, were isolated from aphids in Israel and probably have a similar origin. The pathogenicity ofV. lecanii strains to pupae 6 days after inoculation varied between 59± 12.1 % and 72.5± 13.1 %, as compared with natural mortality of 13.5±4%. The maximum adult mortality caused byV. lecanii strains was between 34.1±5.1% and 52.6±3.8%.

Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.

Diversity among isolates ofVerticillium lecanii as expressed by DNA polymorphism and virulence towardsBemisia tabaci

Thirty-six isolates ofVerticillium lecanii andVerticillium sp. were taken from different hosts (both insects and rusts) and geographical locations. The isolates were analyzed for genomic variability, as expressed by random amplified polymorphic DNA (RAPD), in relation to virulence onBemisia tabaci. Virulence on larvae ofB. tabaci within these isolates ranged from 0% to 83%. RAPD analysis was performed employing two different arbitrary decamer primers and the calculated similarity coefficients were subjected to cluster analysis using the unweighted average linkage (UPGMA) algorithm. The dendrograms obtained with each of the two primers were identical. Eight cluster groups and three unclustered isolates were obtained by selecting a similarity level of 80%. The amplification pattern of DNA obtained by RAPD for the various isolates suggested thatV. lecanii is a highly diverse species. No correlation could be established between virulence and either RAPD polymorphism of the fungal isolates or the insect host from which they were isolated. Generally, no correlation could be established between the clustering ofV lecanii strain and geographical location although a limited number of strains obtained from Russia and Georgia were assembled in the same cluster and those from Kazakhstan were clonal.

Survival of the Nematode-Antagonistic Fungus Verticillium Lecanii in Alginate Prills1,2)

Strains of the fungus Verticillium lecanii have been incorporated into alginate prills and studied as potential management agents for plant-parasitic nematodes. Since long-term storage is essential in material intended for field applications, the viabilities of two V. lecanii strains in prill formulations were recorded after storage in the freezer (-15°C), refrigerator (4°C), ambient room conditions (ca. 25°C), and glasshouse (15-43°C). In a wheat bran formulation, both strains generally grew from 90%-100% of prills storcd in the refrigerator for up to 43-45 months (although one batch of the mutant strain lost viability by 12 months). Viability at -15°C did not always remain as high as when prills were stored at 4°C. Viability of both strains was 0% within 2 years at ambient room conditions and within 7 months in the glasshouse. Similar results were obtained with the mutant strain in a pyrophyllite formulation. One batch of wheat bran prills made with the mutant strain plus soybean cyst nematode sex pheromone (vanillic acid) was also tested. Fungus viability was 0% after 25 months (freezer, refrigerator), 4 months (ambient room conditions), and 1 month (glasshouse). Fungus in prills made with the pheromone analogue syringic acid had a similar longevity to fungus in prills without added pheromone.