Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

A critical review of the application of white rot fungus to environmental pollution control

Research on white rot fungi for environmental biotechnology has been conducted for more than 20 years. In this article, we have reviewed processes for cell growth and enzyme production including the factors influencing enzyme productivity and the methods for enhancement of enzyme production. Significant progress has been achieved in molecular biology related to white rot fungi, especially related to the extraction of genetic material (RNA and DNA), gene cloning and the construction of genetically engineered microorganisms. The development of biotechnologies using white rot fungi for environmental pollution control has been implemented to treat various refractory wastes and to bioremediate contaminated soils. The current status and future research needs for fundamentals and application are addressed in this review.     

Horizontal transmission of the microbial symbionts Enterobacter cloacae and Mycotypha microspora to their firebrat host

The firebrat, Thermobia domestica (Packard) (Thysanura: Lepismatidae), aggregates in response to the faeces of conspecifics. This aggregation response is mediated by two microbial symbionts, the bacterium Enterobacter cloacae (Jordan) Hormaeche & Edwards (Enterobacteriaceae) and the fungus Mycotypha microspora Fenner (Mucorales). Our objective was to determine how these microbes are transmitted between firebrats. We produced fluorescently labelled E. cloacae and M. microspora and presented them to firebrats. Firebrats consumed large quantities of these labelled microbes and deposited them with their faeces where they proliferated rapidly. Firebrats did not harbour E. cloacae or M. microspora within their ovarioles or eggs, and thus cannot transmit them transovarially. Instead, firebrats acquired them horizontally whenever they fed on microbe‐contaminated material, such as faeces, faeces‐contaminated paper, or egg surfaces. Firebrats moult throughout their life, and with each moult they shed the cuticular lining of their digestive tract and likely any microbes residing therein. Because firebrats remain in close contact and live in groups of mixed age and gender, newly moulted individuals can readily re‐acquire E. cloacae or M. microspora from group members. This ensures the perpetuation of their microbial aggregation and arrestment signal.     

丛枝菌根真菌对西瓜嫁接苗生长和根系防御性酶活性的影响

在温室盆栽条件下,研究丛枝菌根(AM)真菌地表球囊霉(Glomus versiforme)对连作土壤中西瓜自根苗和嫁接苗生长、根系膜透性、丙二醛(MDA)含量和防御性酶活性的影响.结果表明： 接种AM真菌能显著增加西瓜自根苗和嫁接苗的生物量,提高根系活力,降低根系膜透性和MDA含量.接种AM真菌的自根苗地上部鲜质量、地上部干质量和根系活力分别增加了57.6%、60.0%和142.1%,而接种AM真菌的嫁接苗分别增加了26.7%、28.0%和11.0%;自根苗（C）、嫁接苗（G）、接种AM真菌自根苗（C+M）和接种AM真菌嫁接苗（G+M）的根系细胞膜透性为C>G>C+M>G+M,根系MDA含量为C>G>G+M>C+M.接种AM真菌能提高西瓜自根苗和嫁接苗根系的苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、过氧化物酶(POD)、几丁质酶和β 1,3 葡聚糖酶活性,而且接种AM真菌的西瓜自根苗和嫁接苗根系POD、PAL和β-1,3-葡聚糖酶活性的峰值比不接种的提前2周出现.接种AM真菌能激活西瓜自根苗和嫁接苗与抗逆性有关的防御性酶反应,使根系对逆境产生快速反应,从而提高其抗连作障碍的能力.     

绵地花菌(Albatrellus ovinus)中的grifolin及其衍生物

本文对高等真菌绵地花(Albatrellus ovinus)进行了化学成分的研究。利用各种柱色谱方法(包括正相硅胶、反相硅胶、Sephadex LH-20凝胶色谱、中压液相色谱、半制备HPLC等),分离得到grifolin及其它的5个衍生物。分别为grifolin(1)、neogrifolin(2)、grifolinone A(3)、grifolinone C(4)、confluentin(5)和4-O-methylgrifolic acid(6)。这些化合物的结构通过波谱学方法以及与文献数据对照进行确定。化合物3～6为首次从该种高等真菌中分离得到。化合物4为一个真菌色素,它是一个由一分子的grifolin和一分子苯醌化的grifolin以头对头的方式聚合而成的二聚体。     

Two strains of Pseudomonas fluorescens bacteria differentially affect survivorship of waxworm (Galleria mellonella) larvae exposed to an arthropod fungal pathogen,Beauveria bassiana

Two strains of Pseudomonas fluorescens were found contaminating a biopesticide used in a previous study against Varroa destructor infestations in honey bee hives. In that study, the biopesticide, a formulation of a fungal pathogen of arthropods, Beauveria bassiana, failed to have any negative impact on the mite infestation despite successful results in previous studies using uncontaminated batches of the same biopesticide. The objective of the present research was to determine whether the bacteria may have interfered with the infectivity and/or virulence of B. bassiana in a simplified system; positive results in that system would then provide a rationale for further work under more complex conditions. Galleria mellonella late instar larvae treated topically with both a bacterial suspension of 6.8 to 7.0×10⁷ cfu/ml and a fungal suspension of 2.5×10⁷ or 2.5×10⁸ B. bassiana conidia/ml showed, in the case of one of the bacterial strains, significantly increased survivorship compared to larvae treated with just the B. bassiana suspension. When larvae were immersed in a bacterial suspension prior to application of B. bassiana suspension using a spray tower, a significant positive effect of the same P. fluorescens strain on larval survivorship was observed at 2.5×10⁸ conidia/ml. Neither the bacterial suspensions alone nor blank control solutions had any effect on larval survivorship. These results show that an interaction between the bacteria and the pathogen may explain some of the results from the prior field trial.     

Post-proline Dipeptidyl Amino-peptidase from Flavobacterium

Nitrogenase catalyzes not only the reduction of N₂ to NH₃ but also the reduction of C₂H₂ to C₂H₄ and H⁺ ion to H₂ gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C₂H₂ reduction activity strongly but did not inhibit H₂ evolution activity. MA-2, on the contrary, inhibited only H₂ evolution activity. MA-8 inhibited both C₂H₂ reduction and H₂ evolution activity to the same extent.     

Isolation and Structure Elucidation of Growth Inhibitors on Silkworm Larvae from Magnolia kobus DC

Two lignans were isolated from leaves of Magnolia kobus DC. as growth inhibitors on silkworm larvae and structurally elucidated as sesamin (I) and kobusin (II) which has been hitherto unknown.