Erratum to: Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions

Doklady Biochemistry and Biophysics - An Erratum to this paper has been published: https://doi.org/10.1134/S1607672922330018     

Carbohydrate Storage in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana was grown in 1% (wt/vol) gelatin-liquid media singly supplemented with a monosaccharide (glucose or fructose), a disaccharide (maltose or trehalose), a polyol (glycerol, mannitol, or sorbitol), or the amino sugar N-acetyl-d-glucosamine. The relative contributions of the carbohydrate, protein, and water contents in the fungal biomass were determined. Carbohydrates composed 18 to 42% of the mycelial dry weight, and this value was lowest in unsupplemented medium and highest in medium supplemented with glucose, glycerol, or trehalose. Biomass production was highest in liquid cultures supplemented with trehalose. When liquid cultures were grown in medium supplemented with 0 to 1% (wt/vol) glucose, trehalose, or N-acetyl-d-glucosamine, there was an increase in the biomass production and the contribution of carbohydrate to mycelial dry weight. Regardless of the glucose concentration in the culture, water content of the mycelia remained about 77.5% (wt/wt). Mycelial storage carbohydrates were determined by capillary gas chromatography. In gelatin-liquid medium supplemented with 1% (wt/vol) glucose, B. bassiana stored glycogen (12.0%, wt/dry wt) and the polyols mannitol (2.2%), erythritol (1.6%), glycerol (0.4%), and arabitol (0.1%). Without glucose, B. bassiana stored glycogen (5.4%), mannitol (0.8%), glycerol (0.6%), and erythritol (0.6%) but not arabitol. To our knowledge, this is the first report of carbohydrate storage in an entomopathogenic fungus, and the results are discussed in relation to other fungi and the potential implications to commercial formulation and insect-fungus interactions.     

Adhesion of the Entomopathogenic Fungus Beauveria (Cordyceps) bassiana to Substrata

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.     

N-Acetyl-d-Glucosamine-Mediated Regulation of Extracellular Protease in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana GK2016 grown in a liquid medium incorporating gelatin as the sole carbon and nitrogen source produced an extracellular serine protease (molecular weight, 35,000; pI ca. 10). Without gelatin, B. bassiana could utilize N-acetyl-d-glucosamine (GlcNAc; 2-acetamido-2-deoxy-d-glucose) as the sole source of carbon and nitrogen, and GlcNAc availability increased the storage carbohydrate content in mycelia. Synthesis of protease was repressed in gelatin medium containing GlcNAc at levels of >1.07 mumol mg of fungal dry weight. At levels below this, protease synthesis was initiated; subsequently, free amino nitrogen appeared in the medium and diauxic growth was observed. Slow feeding with GlcNAc (35.34 mug ml h) did not repress protease synthesis nor did GlcNAc accumulate in the medium above 0.5 mg ml. Increasing the rate of release of GlcNAc (83.51 mug ml h) resulted in the accumulation of GlcNAc in the medium to 2.0 mg ml, a 45% increase in growth and a decrease in protease synthesis by about 81%. Free amino acids generated from the hydrolysis of gelatin did not repress protease synthesis. These data are interpreted in terms of known interaction of B. bassiana with insect cuticular components. We suggest that the action of extracellular chitinases synthesized by B. bassiana on insect cuticle, and pursuant release of GlcNAc, may have important consequences on the regulation of other extracellular catabolic enzymes such as the protease.     

Prevalence and Diversity of Viruses in the Entomopathogenic Fungus Beauveria bassiana

Viruses have been discovered in numerous fungal species, but unlike most known animal or plant viruses, they are rarely associated with deleterious effects on their hosts. The knowledge about viruses among entomopathogenic fungi is very limited, although their existence is suspected because of the presence of virus-like double-stranded RNA (dsRNA) in isolates of several species. Beauveria bassiana is one of the most-studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as a biological control agent against invertebrates in agriculture. We analyzed a collection of 73 isolates obtained at different locations and from different habitats in Spain and Portugal, searching for dsRNA elements indicative of viral infections. The results revealed that the prevalence of viral infections is high; 54.8% of the isolates contained dsRNA elements with viral characteristics. The dsRNA electropherotypes of infected isolates indicated that virus diversity was high in the collection analyzed and that mixed virus infections occurred in fungal isolates. However, a hybridization experiment indicated that dsRNA bands that are similar in size do not always have similar sequences. Particular virus species or dsRNA profiles were not associated with locations or types of habitats, probably because of the ubiquity and efficient dispersion of this fungus as an airborne species. The sequence of one of the most common dsRNA elements corresponded to the 5.2-kbp genome of a previously undescribed member of the Totiviridae family, termed B. bassiana RNA virus 1 (BbRV1).     

Distribution of the Entomopathogenic Fungus Beauveria bassiana in Rice Ecosystems and Its Effect on Soil Enzymes

Fungal entomopathogens, especially Beauveria bassiana, are often studied within the context of their use in biological pest control; however, there is limited knowledge of their distributions in host plants and soil ecosystem. We examined the distribution of B. bassiana and its influence on rice plants and paddy soils. B. bassiana could only be detected on the foliar surfaces of rice plants within 15 days under Bb-4 (7.5 × 10⁴ conidia/mL) and Bb-7 (7.5 × 10⁷ conidia/mL) treatments. The endophytic colonization of B. bassiana could not be found in stems, roots, or seeds of rice plants under Bb-4 and Bb-7 treatments. The fungus was found only in the leaves of rice plants under Bb-4 and Bb-7 treatments at 15 days after inoculation. Moreover, B. bassiana was absent from paddy soils under Bb-4 and Bb-7 treatments at all times. Enzyme activity (urease and phosphatase) in the paddy soils of Bb-4 and Bb-7 treatments showed no significant difference from the control. It is possible that B. bassiana was not able to colonize paddy soil. Detailed understanding of distribution and ecological interactions of B. bassiana is helpful for understanding and predicting the effects of fungal entomopathogens on host populations, and the interactions among fungal entomopathogens and other organisms in the community.     

蚕豆种质资源形态标记遗传多样性分析

通过对国内外不同地理来源637份蚕豆资源的18个形态性状评价,初步了解其遗传多样性特点,寻找蚕豆种质创新与品种改良遗传基础狭窄问题的解决思路,为遗传与育种的有机结合提供理论依据。研究结果表明国内春冬性蚕豆资源间,以及国内外蚕豆资源间的遗传变异大;三维主成分分析探测到参试资源由三大基因库构成。资源群体间和参试资源基因型间遗传距离的UPGMA聚类分析结果,为蚕豆资源的有效利用提供了科学依据。     

Effect of Pre-sowing Magnetic Field Treatment on Some Biochemical and Physiological Processes in Faba Bean (Vicia faba L. spp. Minor)

Journal of Plant Growth Regulation - The aim of this paper was to determine the changes in some biochemical and physiological processes, which occurred in seeds and seedlings of faba bean and to...     

蚕豆种质资源清蛋白遗传多样性分析?

利用SDS-PAGE对101份蚕豆种质资源进行了清蛋白遗传多样性分析,共检测出蛋白带2625条。除共有带外,迁移率不同的谱带类型36种,其中相对分子量为92kD、75kD、62kD、40kD、34kD、17kD、13kD的谱带在检测的种质材料中出现的频率最高,分别为92.08%、90.10%、99.01%、95.05%、95.05%、98.02%、95.05%。其余29种谱带类型具有较强的多态性,多态性谱带平均为16.09条,多态性比例为44.69%。每份种质材料的清蛋白谱带数介于21～31条之间,平均25.99条;供试种质间遗传相似系数0.6111～0.9722,平均0.7122。3个地理类群内多样性指数0.9879,类群间多样性指数0.0121,表明蚕豆清蛋白的变异主要来源于类群内。聚类分析将参试种质分为6类,与以往蚕豆种质分类研究结果类似,表明清蛋白能在一定程度上反映种质间的亲缘关系。研究结果对于蚕豆蛋白质品质育种具有一定的参考价值。     

Horizontal Transmission of the Entomopathogenic Fungus Beauveria bassiana among the Spruce Bark Beetle, Ips typographus (Col., Scolytidae) in the Laboratory and under Field Conditions

In laboratory and field cage experiments, the horizontal transmission of the entomopathogenic fungus Beauveria bassiana (Boverol®) between adults of the spruce bark beetle Ips typographus was examined. In a laboratory experiment, conidia transfer between treated and untreated beetles at different ratios resulted in 96% mortality (MST 4.3 days) at a ratio of 1:1 and 90% mortality (MST 4.5 days) at a ratio of 1:2. At ratios of 1:5, 1:10 and 1:20, the mortalities at 7 days were 83, 77 and 75%, respectively. In a second laboratory experiment, the efficacy of B. bassiana was investigated after transmission from contaminated to healthy beetles over a period of 5 days. Two weeks later, the mortality of treated and untreated beetles was 99% (control mortality was 44%), while the rate of mycosis was 79% in the treatment compared to 10% in the control beetles. A single contact between one treated male (2.0×10⁵ conidia/beetle) and an untreated female was found to be sufficient to transmit a lethal dose of B. bassiana of about 1.2×10⁴ conidia. Two experiments in field cages in a spruce stand demonstrated horizontal transmission of B. bassiana (Boverol®) between beetles. In the first experiment the transmission was investigated following the introduction of B. bassiana-inoculated beetles into a population of untreated beetles. Significant reductions were observed in the length of maternal galleries, and the number of larvae and pupae. In the second field cage experiment, natural populations of beetles were lured into a flight cage using a pheromone trap, and treated with B. bassiana or left untreated on alternate days. In this experiment, significant reductions were observed in the number of bore holes and the length of maternal galleries, while no larvae, pupae or juveniles were found under the bark of spruce trunks in the Boverol® transmission treatment. It is concluded that B. bassiana has potential for control of I. typographus, and further field experiments are warranted to investigate its efficacy under more practical conditions.     

Allee Effect in the Infection Dynamics of the Entomopathogenic Fungus Beauveria bassiana (Bals) Vuill. on the Beetle, Mylabris pustulata

Successful infection by Beauveria bassiana as with all other entomopathogenic fungi, is accomplished only at a high conidial dose while, theoretically, a single conidium should be sufficient. Indeed, this is a major deterrent in its use as a biocontrol agent. High pathogen load for infection is required by organisms which display ‘Allee’ effect. In such organisms, a threshold exists for pathogen dose, below which no infection can be caused. B. bassiana has a semelparous life cycle and, therefore, its infection dynamics are expected to conform to the mass action principle with a linear relationship between dose and successful infection observable as mortality of the insect. Whether the need for a high conidial dose to induce insect mortality by B. bassiana is due to the operation of Allee effect was examined. A sample of 34 isolates was bioassayed on Mylabris pustulata (Coleoptera: Meloidae) at four conidial concentrations. With more than half of the isolates in the sample, the lowest dose tested (10⁴ conidia/insect) did not cause insect mortality. Thus, a threshold pathogen load is required to cause successful infection. In these isolates, the dose–mortality relationship was sigmoid. Allee effect is thus identified in the infection dynamics of B. bassiana–M. pustulata system. The isolates that induced mortality at the lowest dose tested are concluded to be highly virulent with a lower threshold dose required for successful infection. With some isolates, at high conidial dose, the infection rate decreased either due to a decrease in the proportion of insects showing mycosis, to the speed of death, or both. Such a response could result from intra scramble competition arising from overload of pathogen at very high dose.     

Effect of 6-Benzylaminopurine on the Structure of the Photosynthetic Apparatus of Faba Bean (Vicia faba L.)

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (S _max), was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of S _max) and decreased in leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of the terminal phloem; however, it did (1) stimulate elongation of mesophyll cells; (2) increase the area and thickness of the leaf blade, amount of photosynthetic pigments, and assimilation potential; and (3) delay senescence of the leaves and defoliation, thereby increasing the biomass of the aboveground plant part. It is inferred that BAP has the potential for inducing the development of photosynthetic apparatus and increasing the yield of the green mass of faba bean.     

Colonization of Corn, Zea mays, by the Entomopathogenic Fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

The Effect of Reduction in the Number of Nodules on Nodule Activity of Faba Bean (Vicia faba cv. Fiord)

Faba bean (Vicia faba L. cv. Fiord) plants were raised in agrowth room for 5 weeks and then transplanted to a hydroponicsystem. After 48 h for acclimation, nine plants were removed(day 0) for the measurement of nitrogenase activity by acetylenereduction (AR), for determination of nodule number and noduleweight, volume of the active N₂ fixing region (VAR), and volumeof the senescent N₂, fixing region (VSR). Half the nodule populationon a further 18 plants was excised, and nine of these plantswere assayed for AR. The nine plants from which the noduleshad been removed (treated plants) and nine control plants witha full complement of nodules, were left to grow for 5 d, afterwhich they were all harvested and assayed. The average weight of nodules and VAR remained constant in thecontrol plants between day 0 and day 5, whereas the nodulesleft on the treated plants increased in weight by 1.2 timesand VAR by 2.2 times. By day 5, VAR per plant was the same inthe control plants as in the treated, whereas VSR of controland treated plants increased by 4.6 and 2.2 times, respectively.Removal of half of the nodules at day 0 halved the AR activityper plant, but specific activity remained the same. After 5d, however, the nodules of the treated plants showed the sametotal activity as those of the control. Thus the specific activityof the nodules left on the treated plants doubled after 5 din response to excision. The indeterminate nodule of faba bean appears to be able toincrease its specific activity substantially in response toincrease in the demand for fixed N. N₂ fixation per nodule wasresponsive to substrate supply, in that halving the number ofnodules on a plant induced the remaining nodules to increaseactivity, presumably because they could use the assimilate previouslydistributed over a larger number of nodules. Victa faba, faba bean, nodule number, nodule activity, acetylene reduction, volumes of active and senescent N₂ fixing regions of nodules, hydroponic system     

繁殖群体量及隔离对蚕豆种质遗传完整性的影响

为研究繁殖群体量和隔离方式对常异花授粉作物蚕豆种质繁殖更新的影响,以9份蚕豆地方种质为对象,以国家库保存的原种为对照群体,采用AFLP分子标记方法,对比了50株和20株群体量及开放无隔离群体和网棚隔离群体更新后代的遗传完整性差异。结果表明:与对照群体相比,50株和20株群体的多态性位点数、多态位点百分率、每位点有效等位基因数、香农指数、遗传多样性指数均出现不同程度下降,但下降幅度20株大于50株群体;遗传相似性和UPGMA聚类分析表明50株群体与对照群体的遗传相似性高于20株群体;网棚隔离可降低群体间串粉与花粉污染,但其遗传完整性却较开放无隔离群体低。     

Photothermal Time for Flowering in Faba Bean (Vicia faba) and the Analysis of Potential Vernalization Responses

One cultivar and one land-race of faba bean were subjected to18 potentially vernalizing pre-treatments (constant temperaturesof 1, 5 or 9 °C factorially combined with photoperiods of8 or 16 h d^–1 for 10, 30 or 60 d), and then transferredinto four different growing regimes (‘day’/‘night’temperatures of 18/5 °C or 24/13 °C factorially combinedwith photoperiods of 11 or 16 h d–1). Control plants weregrown entirely in the latter four regimes. The times from sowingto appearance of first open flowers were recorded for all plants.Control plants of the land-race Zeidab Local flowered soonerin long days and in the warmer regime. Pre-treatment reducedthe subsequent time to flower in the four growing-on regimesbut most of the variation in the total time to first flowerfor the pre-treated plants was accounted for by differencesin the combined photothermal time accumulated in the two successiveenvironments - which was predicted by a simple photothermalmodel. Thus, there was neither a specific low-temperature nora short-day vernalization response in this accession. Similarly,no true low-temperature or short-day vernalization responsewas detected in the cv. Maris Bead. However, this UK cultivarflowered later than predicted in the 24/13 °C regime, indicatingthat the 24 °C ‘day’ temperature was supraoptimal.Delays to flowering at 24/13 °C were, however, less evidentwhen plants were grown in long days or following prolonged (30–60d) pre-treatments at cool temperatures. Viciafaba faba, bean, flowering, photoperiodism, vernalization, photothermal time, screening germplasm.     

Reduction of Banana Weevil Populations Using Different Formulations of the Entomopathogenic Fungus Beauveria bassiana

One of the major constraints for banana production in Uganda is the banana weevil, Cosmopolites sordidus (Germar), (Coleoptera: Curculionidae). Investigations were carried out to evaluate the efficacy of maize, soil-based and oil formulations of an indigenous isolate of Beauveria bassiana for the control of the banana weevil. Weekly trapping of weevils over a 9-month monitoring period showed significant reduction in unmarked and marked weevil population in B. bassiana treated plots. Application of maize formulation at 2 ×10 15 conidia ha -1 proved most effective, reducing the weevil populations by 63-72% within 8 weeks after a single application. The soil based formulation at 2 ×10 14 conidia ha -1 was intermediate while the oil formulation at 6 ×10 15 conidial ha -1 was least effective. Trapping efficiency declined in B. bassiana treated and untreated banana plots but was greatest in the latter.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.