Bombus terrestris as pollinator-and-vector to suppress Botrytis cinerea in greenhouse strawberry.

BACKGROUND: Bombus terrestris L. bumblebees are widely used as commercial pollinators, but they might also be of help in the battle against economically important crop diseases. This alternative control strategy is referred to as pollinator-and-vector technology. The present study was designed to investigate the capacity of B. terrestris to fulfil this role in greenhouse strawberry flowers, which were manually inoculated with a major plant pathogen, the grey mould Botrytis cinerea Pers.: Fr. A model microbiological control agent (MCA) product Prestop-Mix was loaded in a newly developed two-way bumblebee dispenser, and, in addition, the use of the diluent Maizena-Plus (corn starch) was tested. RESULTS: Importantly, loading of the MCA caused no adverse effects on bumblebee workers, with no loss of survival or impairment of flight activity of the workers during the 4 week flowering period. Secondly, vectoring of Prestop-Mix by bumblebees resulted in a higher crop production, as 71% of the flowers developed into healthy red strawberries at picking (preharvest yield) as compared with 54% in the controls. In addition, these strawberries were better protected, as 79% of the picked berries remained free of B. cinerea after a 2 day incubation (post-harvest yield), while this percentage was only 43% in the control. Overall, the total yield (preharvest × post-harvest) was 2-2.5 times higher than the total yield in the controls (24%) in plants exposed to bumblebees vectoring Prestop-Mix. Thirdly, the addition of the diluent Maizena-Plus to Prestop-Mix at 1:1 (w/w) resulted in a similar yield to that of Prestop-Mix used alone, and in no negative effects on the bumblebees, flowers and berries. CONCLUSIONS: This greenhouse study provides strong evidence that B. terrestris bumblebees can vector a MCA to reduce B. cinerea incidence in greenhouse strawberries, resulting in higher yields. Similar yields obtained in the treatments with Prestop-Mix and Prestop-Mix + Maizena-Plus suggest an equally efficient dissemination of the biocontrol agent into the flowers with only half the initial concentration of Prestop-Mix, which illustrates the importance of the diluent.

Compatibility between Clonostachys isolates with a view to mixed inocula for biocontrol.

To aid the development of compatible biocontrol inocula, a prescreening method for the prediction of compatibility of fungal antagonists was developed. Compatibility between 18 Clonostachys isolates with known antagonistic capabilities against Phytophthora palmivora was tested using intra- or interisolate pairings (dual cultures) on water agar plates, a hyphal interaction experiment and a modified double host-range experiment. Almost all inter- or intraisolate pairings of Clonostachys isolates showed growth inhibition zones and did not show free hyphal intermingling. A hyphal interaction experiment on water agar demonstrated that the aggressiveness of a Clonostachys isolate and its susceptibility to mycoparasitism were unrelated phenomena. However the level of aggressiveness and/or susceptibility of an isolate were largely dependant on the isolate with which it was challenged. The degree of growth-inhibition caused by an isolate was unrelated to the hyphal damaged it caused or received. In the double host-range experiment all possible pairs from four Clonostachys isolates were inoculated in different ratios (10 000-fold range) on plates precolonized with one of two P. palmivora isolates. The results showed that antagonistic capabilities of certain combinations were affected by the Clonostachys isolates. The primary host, P. palmivora, did not affect antagonistic capabilities; whereas inoculum ratio did. Of note, it was not possible to predict the outcome of the double host range on the basis of the results of the hyphal interaction experiment. In conclusion the competitive abilities of Clonostachys isolates depend on the partner with which they are applied and less on resource availability. The double host-range test as developed here might provide the most representative tool to date to test compatibility of fungal antagonists to be used in biocontrol inocula. However the link between the results of the double host-range test and field efficacy of biocontrol inocula remains to be investigated.

Evaluation of the effects of chemical versus biological control on Botrytis cinerea agent of gray mould disease of strawberry.

This study investigates on effects of four fungicide and six isolate from Trichoderma and Gliocladium on Botrytis cinerea agent grey mold of strawberry under library and greenhouse condition. The effect of four fungicides i.e. benomyl, dichlofluanid, captan and triadimenol on B. cinerea was studied in the laboratory condition by method mixed poison to culture medium. It was shown that the fungicide including benomyl, triadimenol, dichlofluanid and captan were able to inhibit mycelial growth of B. cinerea on PDA plate with EC50 of 0.16, 1.42, 3.40 and 7.73 ppm respectively. These fungicides delayed myceliogenic germination of sclerotia at 1000 ppm, while exhibiting no fungicidal effect. Moreover, the antagonistic effects of six fungi including Trichoderma koningii (T21), T. viride (T4), T. harzionum (T5), T. viride (T2), G. virens (G2), G. virens (G8) on B. cinerea were assessed. This assessment was done under library condition and its results as follows: The antagonistic mechanism occurred through branching at the end of B. cinerea hyphae, hyphal contact, coiling, vacuolization and lyses. Volatile metabolites of T. koningii (T21) and non-volatile metabolites of G. virens (G2 and G8) and T. koningii (T21) caused maximum inhibition of the fungal growth. Trichoderma spp and G. virens were able to colonize and sporulate on sclerotia and caused their lysis within 7-21 days. In greenhouse, a completely randomized design with 11 treatments (4 chemical and 6 biological and one untreated control) each replicated five times were used for the comparison. Greenhouse studies revealed that application of fungicides i.e. captan, dichlofluanid, triadimenol and benomyl reduces disease severity by 42, 45, 48 and 52% respectively. The fungal antagonists reduce the grey mold disease severity between 5-42%. All treatments caused a decline in post harvest disease, as the most effective treatment of chemical control was benomyl with 68.33% and for the biological treatment this was T. koningii (T21) with 56%.

[Allelopathy of root exudates from two genotypes soybeans on root pathogenic fungi].

With biological simulation experiment and chemical analysis, this paper studied the allelopathy of carbohydrates, amino acids and organic acids in the root exudates from two genotypes soybeans (9536 and Jilin 30) on the pathogenic fungi of root rot. The results showed that the water soluble carbohydrates in the root exudates from test soybeans significantly promoted the growth of Fusarium oxysporium and Fusarium semitectum at low concentrations and inhibited their growth at high concentrations, but had no evident influence on Gliocladium roseum. The water soluble amino acids from the root exudates demonstrated different actions, i. e., at middle and high concentrations, those from 9536 significantly inhibited the growth of Fusarium oxysporium, Fusarium semitectum and Gliocladium roseum, while those from Jilin 30 mostly promoted their growth. The organic acids from the root exudates of 9536 and Jilin 30 significantly inhibited the growth of Fusarium oxysporium, Fusarium semitectum and Gliocladium roseum. It's suggested that there existed interactions between the root exudates of the two genotypes soybeans and the pathogenic fungi of root rot. Different genotypes of soybean may have different allelopathy on pathogenic fungi of root rot.

Efficacy of microorganisms antagonistic to Rhizoctonia cerealis and their cell wall degrading enzymatic activities.

The effect of Trichoderma atroviride, T. harzianum, T. longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions. Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control. Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp. Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied. Almost all tested antagonists showed competitive activity against R. cerealis: inhibition of its mycelial growth and hyphal interaction. The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R. cerealis was then determined. All isolates showed glucosaminidase and chitobiosidase activity. They also produced glucosidase activity, except B. subtilis, whereas only C. rosea, B. subtilis and one isolate of T. harzianum showed detectable levels of protease activity.

Induction of differentiation in acute promyelocytic leukemia cells (HL-60) by the verticillin derivative Sch 52900.

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, is a widely used model system to study the cellular and molecular events involved in differentiation of leukemic cells. In a screening for inducers of differentiation of HL-60 cells, cultures of Gliocladium strain 4-93 were found to produce Sch 52900, a previously isolated diketopiperazine (Chu et al,. J Antibiotics 48, 1440-1445). Sch 52900 induced the differentation of 50-69% of HL-60 cells at concentrations of 6.8-13.6 nM as measured by nitro-blue tetrazolium chloride (NBT) reduction which was followed by apoptosis as shown by DNA fragmentation. Our results demonstrate that growth arrest and the induction of differentiation by Sch 52900 is due to the induction of the cell cycle inhibitor p21WAF and an inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway which leads to the activation of the transcription factor AP-1.

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists.

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.

In vitro evaluation of trichoderma and gliocladium antagonism against the symbiotic fungus of the leaf-cutting ant Atta cephalotes.

The antagonistic activity of Trichoderma and Gliocladium isolates against Attamyces sp., a symbiotic fungus of the leaf-cutting ant Atta cephalotes, was investigated. A. cephalotes cultures this fungus as the primary food source. Most of the Trichodema and Gliocladium isolates tested in vitro (82.6%) inhibited the Attamyces sp. mycelial growth. which was probably due to their colonization ability and competition for nutrients, both of them known mechanisms of some species of these genera. T. lignorum strain T-26 was the strongest inhibitor achieving a colonization of 23%. Microscopical observations indicate that the inhibitory effect was caused by an interaction that took place in close contact with the host hypha, causing wall deformation that led to the collapse of the turgor pressure.

[Secondary metabolites of Gliocladium sp., a growth accelerating fungus for Anoectochilus roxburghii (Wall.) Lindl].

OBJECTIVE: To study the secondary metabolites of fungus Gliocladium sp. that helps accelerate the growth of A. roxburghii. METHOD: Compoud isolation by chromatography and structure elucidation by chemical and spectral analyses. RESULTS: Five compounds were obtained and elucidated as: 8(E)-N-(2'-hydroxypalmityl)-1-O-beta-gly-copyranosyl-3-hydroxyl-9-methyl-2- octodecanine-4, 8-diene (I), N-(2'-hydroxytetracosanoyl)-1,3,4-trihydroxy-2-octodecanine(II), 7, 22-diene-3-hydroxy-6,9-epidioxyergosta(III), ergostol(IV) and alpha-palmitin(V). CONCLUSION: I, II, III were obtained from Gliocladium sp. for the first time.