Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth,Galleria mellonella

The effects of Metarhizium anisopliae infection and three different secondary metabolites released by the fungus, destruxin A and E and cytochalasin D, on the morphology and cytoskeleton of plasmatocytes of the greater wax moth Galleria mellonella were studied. Plasmatocytes isolated from M. anisopliae infected larvae exhibited impairment of attachment, spreading and cytoskeleton formation accompanied with the occurrence of blebbing and pycnotic nuclei. Plasmatocytes treated with destruxin in vitro exhibited similar morphological and cytoskeleton alterations. The corresponding effects were characterized by inhibition of attachment, spreading and filopodia formation as well as by impaired formation of actin filaments and microtubules. Cytochalasin was shown to affect plasmatocytes in vitro in a different manner than destruxin A and E. The results of our comparative study strongly suggested that the morphology and cytoskeleton alterations of plasmatocytes observed in M. anisopliae infected larvae were predominantly caused by destruxins released by the fungus during mycosis. Its mode of action is discussed with regard to present knowledge about its effects on target cells.     

Circular dichroism analysis of destruxins from Metarhizium anisopliae

Destruxins are secondary metabolites secreted by Metarhizium anisopliae [Y. Kodaira, Toxic substances to insects, produced by Aspergillus ochraceus and Oopsra destructor, Agric. Biol. Chem., 25 (1961) 261-262. D.W. Roberts, Toxins from the entomogenous fungus Metarhizium anisoplaie: Isolation from submerged cultures, J. Invertebr. Pathol., 14 (1969) 82-88. D.W. Roberts, Toxins from the entomogenic fungi in microbial control of pest and plant disease, Academic press, New York, 1981, pp441-464.]. In recent research, other than being used as insecticides, destruxins exhibited great potential in therapeutical applications such as antitumor, antivirus, and animal cell immunization effectiveness, etc. In this study, the conformations purified destruxins were determined by circular dichroism (CD). The results indicated that these cyclic peptides have the type I beta-turn conformation. In addition, different types of destruxins exhibited different CD spectra in acetonitrile. Therefore, these characters can be used as fingerprints to identify each type of destruxin. To further investigate the interactions among destruxins, various combinations of destruxins in 10 mM phosphate-buffered saline (PBS) were also studied by CD. The results strongly suggested that destruxins might work independently in vivo. To our knowledge, this is the first report presenting the CD analysis of purified destruxins.     

The destruxins: synthesis, biosynthesis, biotransformation, and biological activity

Destruxins, secondary metabolites first reported in 1961, are cyclic hexadepsipeptides composed of an alpha-hydroxy acid and five amino acid residues. The name "destruxin" is derived from "destructor" from the species Oospora destructor, the entomopathogenic fungus from which these metabolites were first isolated. Individual destruxins differ on the hydroxy acid, N-methylation, and R group of the amino acid residues; where established, the configurations of the amino acid residues are S, and those of the hydroxy acids are R. Destruxins exhibit a wide variety of biological activities, but are best known for their insecticidal and phytotoxic activities. The great interest in destruxins derives from their potential role as virulence factors in fungi, whether such microorganisms are useful insect biocontrol agents or detrimental, causing great plant disease epidemics. Reports on isolation, chemical structure determination, total synthesis, transformation by diverse organisms, and biological activity of destruxins and related metabolites are reviewed for the first time.     

A new destruxin as inhibitor of vacuolar-type H+-ATPase of Saccharomyces cerevisiae

In the course of our screening efforts to discover small molecules as selective inhibitors of vacuolar-type H+-ATPase of Saccharomyces cerevisiae, we have identified eight active destruxins, 1-8, from the fungus Metarhizium anisopliae. The structures were elucidated by extensive 1D- and 2D-NMR spectroscopy, and MS spectrometry. One of these compounds, 8, a regioisomer of chlorohydrin destruxin E (7), is a new destruxin.     

感染水椰八角铁甲的绿僵菌的分子鉴定及致病力测定

在室内饲养的水椰八角铁甲Octodonta nipae(Maulik)种群中,发现有大量甲虫被病原菌感染致死.对死虫体表的病原真菌进行分离鉴定,并依据ITS序列分析鉴定,确定该病原真菌为金龟子绿僵菌小孢变种(Metarhizium anisopliae var.anisopliae).经室内致病力测定,接种浓度分别为1...     

Destruxin from Metarhizium anisopliae induces oxidative stress effecting larval mortality of the polyphagous pest Spodoptera litura

Nine‐day‐old Spodoptera litura (Fab.) larvae were treated with crude destruxin (dtx) extracted from a high‐virulent (M‐19) and a low‐virulent (M‐10) isolate of the entomopathogenic fungus, Metarhizium anisopliae (Metch.), at doses that caused 30%, 50% and 90% mortality in the treated groups after 48 h. Destruxins produced a dose‐dependent decrease in the body weight of the larvae after 1, 24 and 48 h of treatment. There was a considerable hike in the activity of lipoxygenase and lipid peroxidation levels in the treated larvae with increased time of exposure to mycotoxin. The activities of total superoxide dismutase, total catalase, total peroxidase and specific ascorbate peroxidase in the larval body also registered alterations in the dtx‐treated larvae, suggesting that exposure of larvae to crude dtx induces oxidative stress which is countered by the antioxidant enzymes to an extent governed by the concentration and time of treatment, beyond which the larvae succumb to the ecofriendly biotoxin.     

Destruxin production by the entomogenous fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> in insects and factors influencing their degradation

There is much public interest in the use of fungal biological control agents as alternatives to chemical pesticides. However, there are some concerns as to whether the metabolites produced by these fungi pose a risk to humans and the environment. Destruxins are the main metabolites produced by the insect pathogenic fungus Metarhizium anisopliae (Metsch.) Sorok. The production of these compounds in two different insect hosts and their subsequent fate in the soil were assessed as a case study. Destruxin profiling revealed that the amount and type of destruxin produced was dependant upon the fungal strain and insect host and that these compounds decomposed shortly after host death. Destruxin decomposition was presumably due to the activity of hydrolytic enzymes in the cadavers and appeared to be independent of host or soil type and biota. Temperature strongly influenced destruxin decomposition. Our studies are the first to show that the destruxins are essentially restricted to the host and pathogen and are unlikely to contaminate the environment or enter the food chain.     

Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor

Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in DeltaManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in DeltaManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. DeltaManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the DeltaManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.     

绿僵菌素的分离制备及其对蛴螬的毒力

以金龟子绿僵菌金龟子变种Metarhizium anisopliae var. anisopliae菌株MaQ10发酵液为材料,采用萃取、浓缩、制备色谱及重结晶技术,分离纯化出5种绿僵菌素的晶体,经与标准样品以及参考文献相对照,此5种晶体与绿僵菌素A、A2、B、C和E相吻合。进而,采用浸渍法测定了绿僵菌素A和B对大等鳃金龟Exolontha serrulata (Gyllenhall) 和卵圆齿爪鳃金龟Holotrichia ovata Chang 1龄幼虫的触杀毒力。结果表明： 绿僵菌素A和B对大等鳃金龟的触杀活性,在处理后96～120 h内最高,LC₅₀分别为78.1571 mg/L和88.7562 mg/L; 处理浓度为300 mg/L时,LT₅₀分别为13.4159 h和10.5331 h。绿僵菌素A和B对卵圆齿爪鳃金龟的触杀活性,在处理后96 h时最高,LC₅₀分别为66.5308 mg/L和79.4309 mg/L;处理浓度为300 mg/L时,LT₅₀分别是13.6399 h和9.9451 h。     

Water content and water activity for the production of cyclodepsipeptides in solid-state fermentation by Metarhizium anisopliae

Metarhizium anisopliae was grown by solid-state fermentation on a series of compositions of rice, rice bran, or rice husk media for the production of cyclodepsipeptides, destruxins A and B. The best yield for destruxin A and destruxin B were 2.9 mg kg^–1 substrate and 227 mg kg^–1 substrate, respectively, after 14 days cultivation with rice/bran/husk medium at 71% water content together with water activity of 0.921.     

Effectiveness of Bacillus thuringiensis-transgenic chickpeas and the entomopathogenic fungus Metarhizium anisopliae in controlling Helicoverpa armigera (Lepidoptera: Noctuidae)

The use of genetically modified (Bt) crops expressing lepidopteran-specific Cry proteins derived from the soil bacterium Bacillus thuringiensis is an effective method to control the polyphagous pest Helicoverpa armigera. As H. armigera potentially develops resistance to Cry proteins, Bt crops should be regarded as one tool in integrated pest management. Therefore, they should be compatible with biological control. Bioassays were conducted to understand the interactions between a Cry2Aa-expressing chickpea line, either a susceptible or a Cry2A-resistant H. armigera strain, and the entomopathogenic fungus Metarhizium anisopliae. In a first concentration-response assay, Cry2A-resistant larvae were more tolerant of M. anisopliae than susceptible larvae, while in a second bioassay, the fungus caused similar mortalities in the two strains fed control chickpea leaves. Thus, resistance to Cry2A did not cause any fitness costs that became visible as increased susceptibility to the fungus. On Bt chickpea leaves, susceptible H. armigera larvae were more sensitive to M. anisopliae than on control leaves. It appeared that sublethal damage induced by the B. thuringiensis toxin enhanced the effectiveness of M. anisopliae. For Cry2A-resistant larvae, the mortalities caused by the fungus were similar when they were fed either food source. To examine which strain would be more likely to be exposed to the fungus, their movements on control and Bt chickpea plants were compared. Movement did not appear to differ among larvae on Bt or conventional chickpeas, as indicated by the number of leaflets damaged per leaf. The findings suggest that Bt chickpeas and M. anisopliae are compatible to control H. armigera.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

黄绿绿僵菌对褐飞虱和白背飞虱不同发育阶段的病原性的研究

本文报道了不同孢子浓度下黄绿绿僵菌对褐飞虱和白背飞虱不同发育阶段的易感性和毒力的研究。实验设10.5孢子/mm2,116.7孢子/mm2和1027.1孢子/mm2三种孢子剂量,两种飞虱分为幼龄若虫(1龄和2龄若虫)、高龄若虫(3、4、5龄若虫)和成虫三个发育阶段。实验发现褐飞虱与白背飞虱的三个发育阶段对黄绿绿僵菌的不同浓度的孢子液有不同程度的易感性。黄绿绿僵菌对褐飞虱幼龄若虫的毒力指标LT50在三种孢子剂量下依次为>21、20.82和16.55;对高龄若虫的LT50在三种孢子剂量下依次为17.68、15.49和13.98;而对成虫的LT50在三种孢子剂量下依次为17.10、12.57和9.14。黄绿绿僵菌对白背飞虱幼龄若虫的毒力指标LT50在三种孢子剂量下依次为>21、17.29和13.13;对高龄若虫的LT50在三种孢子剂量下依次为16.94、15.02和13.03;而对白背飞虱成虫的LT50在三种孢子剂量下依次为12.78、10.16和7.64。二者的成虫的易感性比若虫的易感性强,高龄若虫的易感性比幼龄若虫的强。白背飞虱比褐飞虱对黄绿绿僵菌更加敏感。二者的死亡率随孢子浓度的增大而增大。     

The Effect of 2-Chloroethyltrimethyl Ammonium Chloride (CCC) on Stomatal Diffusive Resistance in Tomato

CCC (2-chloroethyltrimethyl ammonium chloride) at a concentration of 6.3 mM was applied to tomato plants (cv. Grosse Lisse) grown in a controlled environment. There was an increase in adaxial leaf diffusive resistance but not in abaxial resistance, the effect being apparent before any growth retardation was measurable. The partial closure of adaxial stomata in response to CCC reduced transpiration from that leaf surface. In plants deprived of water, leaf water potential was higher when CCC was applied and both adaxial and abaxial stomatal closure was delayed. The data do not suggest that CCC influenced the relationship between leaf water potential and conductance for either abaxial or adaxial stomata.     

Leaf Water Repellency as an Adaptation to Tropical Montane Cloud Forest Environments

Adaptations that reduce water retention on leaf surfaces may increase photosynthetic capacity of cloud forests because carbon dioxide diffuses slower in water than air. Leaf water repellency was examined in three distinct ecosystems to test the hypothesis that tropical montane cloud forest species have a higher degree of leaf water repellency than species from tropical dry forests and species from temperate foothills-grassland vegetation. Leaf water repellency was measured by calculating the contact angle of the leaf surface and the line tangent to a water droplet through the point of contact on the adaxial and the abaxial surface. Leaf water repellency was significantly different between the three study areas. The hypothesis that leaf water repellency is higher in cloud forest species than tropical dry forests and temperate foothills-grassland vegetation was not confirmed in this study. Leaf water repellency was lower for cloud forest species (adaxial surface = 50.8°; abaxial surface = 82.9°) than tropical dry forest species (adaxial surface = 74.5°; abaxial surface = 87.3°) and temperate foothills-grassland species (adaxial surface = 77.6°; abaxial surface = 95.8°). The low values of leaf water repellency in cloud forest species may be influenced by presence of epiphylls and loss of epicuticular wax on the leaf surfaces.     

Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection

Although anthocyanin coloration in lower (abaxial) leaf cells has been documented for numerous species, the functional significance of this character has not been comprehensively investigated according to habitat or leaf orientation. Here, we demonstrate that abaxial anthocyanin may function as a photoprotectant, similarly to its purported role in upper (adaxial) cells, in leaves vulnerable to high irradiance incident on abaxial surfaces. Spectral scans were derived for Galax urceolata leaves with the following phenotypes: abaxial or adaxial anthocyanin only, abaxial and adaxial anthocyanin, and no anthocyanin. To determine whether anthocyanins conferred protection from photoinhibition, maximum photosystem II efficiencies of red (anthocyanic) and green (acyanic) surfaces were compared during and after exposure to photoinhibitory conditions. Leaves were either positioned with their adaxial surfaces facing the light source or inverted to expose abaxial surfaces. Spectral scans showed increased absorptance of 500-600 nm wavelengths by red surfaces (consistent with the absorbance spectrum of anthocyanin), regardless of whether that surface was abaxial or adaxial. Leaves with anthocyanin in either illuminated surface were also photoinhibited less than leaves lacking anthocyanin in that surface. These results suggest that anthocyanic layers reduce absorbed sunlight in the mesophyll not only for adaxial surfaces, but also for the abaxial. Adaxial/abaxial anthocyanin plasticity may therefore be adaptive in high-light environments or during light-sensitive developmental stages where leaf orientation and/or substrate albedo are variable.     

Distinct light responses of the adaxial and abaxial stomata in intact leaves of Helianthus annuus L

Using a laboratory-constructed system that can measure the gas exchange rates of two leaf surfaces separately, the light responses of the adaxial and abaxial stomata in intact leaves of sunflower ( Helianthus annuus L.) were investigated, keeping the intercellular CO₂ concentration ( C _i) at 300  µ L L⁻¹. When evenly illuminating both sides of the leaf, the stomatal conductance ( g _s) of the abaxial surface was higher than that of the adaxial surface at any light intensity. When each surface of the leaf was illuminated separately, both the adaxial and abaxial stomata were more sensitive to the light transmitted through the leaf (self-transmitted light) than to direct illumination. Relationships between the whole leaf photosynthetic rate ( A _n) and the g _s for each side highlighted a strong dependence of stomatal opening on mesophyll photosynthesis. Light transmitted through another leaf was more effective than the direct white light for the abaxial stomata, but not for the adaxial stomata. Moreover, green monochromatic light induced an opening of the abaxial stomata, but not of the adaxial stomata. As the proportion of blue light in the transmitted light is less than that in the white light, there may be some uncharacterized light responses, which are responsible for the opening of the abaxial stomata by the transmitted, green light.     

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.     

PATHOGENICITY OF METARHIZIUM ANISOPLIAE VAR. ACRIDU TO THE DEVEOLPMENTAL STAGES OF BROWN PLANTHOPPER NILAPARVATA LUGENS STÅL AND SOGATELLA FURCIFERA (HORVATH)

Abstract  The susceptibility and virulence of entomopathogenic fungus Metarhizium anisopliae var. acridum to the stages of brown planthopper (BPH), Nilaparvata lugens (Stål) and whitebacked planthopper (WBPH), Sogatella furcifera (Horvath) were investigated under laboratory conditions. Three dosages of M. anisopliae var. acridum ranging from 10.5, 116.3 and 1027.1 conidial/mm² were used in the experiment. The tested stages of host included three developmental stages, young nymphs (1–2 instars), old nymphs (3–5 instars) and adults. It was found that all tested stages of the planthoppers were susceptible to the fungal infection. The degree of virulence LT₅₀ of M. anisopliae var. acridum against young nymphs of N. lugens are >21, 20.82 and 16.55, respectively with the 3 dosages, the LT₅₀ of the fungus against the old nymphs are 17.68, 15.49 and 13.98, respectively with the 3 dosages; the LT₅₀ of the fungus against the adults are 17.10, 12.57 and 9.14 respectively with the 3 dosages. The degree of virulence LT₅₀ of M. anisopliae var. acridum on young nymphs of S. furcifera are >21, 17.29 and 13.13, respectively with the 3 dosages _; the LT₅₀ of the fungus against the old nymphs are 16.94, 15.02 and 13.03, respectively with the 3 dosages; the LT₅₀ of the fungus against the adults are 12.78, 10.16 and 7.64, respectively with the 3 dosages. Adults were more susceptible to M. anisopliae var. acridum infection than their nymphs and the young nymphs were most resistant to the fungal infection. The cumulative mortality of each stage was dosage-dependent. Of all the developmental stages, WBPH was more susceptible than BPH to M. anisopliae var. acridum infection with the same dosages.     

Ontogenetic changes in stomatal size and conductance of sunflowers

The ontogenetic changes in stomatal size, frequency and conductance (g_s) on abaxial and adaxial leaf surfaces of sunflower plants (Helianthus annuus L. Russian Mammoth) were examined under controlled environmental conditions. The stomatal frequency on the adaxial and abaxial leaf surfaces decreased with leaf ontogeny and insertion level. The ratio of adaxial to abaxial stomatal frequency did not change with leaf ontogeny and insertion level, and 42–44% of total stomata was apportioned to the adaxial surface. Ontogenetic changes in stomatal pore length were detected and increased with ontogenesis. The stomatal length of both leaf surfaces had linear relationships with leaf area. Ontogenetic changes in g_s were similar between the two surfaces. However the adaxial g_s was lower than abaxial g_s in leaves of higher insertion levels. Conductance had a linear relationship with width x frequency but not with pore area.