Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Aims: Metarhizium anisopliae is an entomopathogenic fungus (EPF) that exists naturally in the environment and potentially be used as a biological control agent against many insect pests. This study aims to evaluate the effect of nutrient additives on the yield and viability of M. anisopliae spore and to determine the optimum incubation period for maximum spore production. Methodology and results: In this study, M. anisopliae was cultivated by solid-state fermentation using rice as a growth medium. Three different nutrient additives were examined which aimed to maximize the production of M. anisopliae spores. Among the three nutrient additives evaluated, yeast (1.84 ± 0.04 g) supported better growth and spore production than molasses (0.58 ± 0.04 g) and palm oil (0.47 ± 0.09 g). The incubation period between 2-6 weeks produced higher spore yield (0.97 ± 0.02 g spores) at week 4 with a better spore viability (86.30 ± 0.45%) at week 2. Conclusion, significance and impact of study Hence, it is suggested that the optimum incubation period is between 2 and 6 weeks after inoculation, and M. anisopliae could be mass produced in large quantities on rice substrate with the addition of yeast as the nutrient additives.
Biological Control Agents ; Microbial Viability ; Metarhizium

Aims: Cruciferae crops failure such as cabbage generally due to heavy attacks by of Crocidolomia pavonana larvae. Metarhizium anisopliae is a species of fungus that can infect more than 200 insect pests including C. pavonana larvae. However, the direct application of M. anisopliae spore in the field is susceptible to UV rays from the sun, which can decrease the efficiency of M. anisopliae. Thus, this study aims to investigate the encapsulation performance of M. anisopliae spore with zeolite and magnesium silicate nanoparticles in terms of mortality and lethal time against 3 rd instar C. pavonana larvae. Methodology and results: Zeolite and magnesium silicate nanoparticles were chosen because they are non-toxic, environmentally friendly and able to maintain moisture, so that they are best used as protective materials for spores. The experiment was designed by Randomized Block Design (RBD) with a single factor. The obtained results showed that encapsulation of M. anisopliae spores with zeolite nanoparticles coating agent increased the mortality rate to 92.5% and accelerated the lethal time up to 1.075 days compared to only spore correspondingly 27.5% and 2.235 days. The M. anisopliae spores encapsulated with magnesium silicate nanoparticles also increased the mortality to 85.0% and accelerated the larval lethal time up to 1.150 days. Conclusion, significance and impact of study Metarhizium anisopliae spores that encapsulated with zeolite nanoparticles coating received higher mortality and faster lethal time to C. pavonana compared to those encapsulated with magnesium silicate nanoparticles. The encapsulation formulation of these two coatings can be used as bioinsecticide in controlling C. pavonana larvae.
Metarhizium

The green peach aphid (Myzus persicae), a plant pest, and gray mold disease, caused by Botrytis cinerea, affect vegetables and fruit crops all over the world. To control this aphid and mold, farmers typically rely on the use of chemical insecticides or fungicides. However, intensive use of these chemicals over many years has led to the development of resistance. To overcome this problem, there is a need to develop alternative control methods to suppress populations of this plant pest and pathogen. Recently, potential roles have been demonstrated for entomopathogenic fungi in endophytism, phytopathogen antagonism, plant growth promotion, and rhizosphere colonization. Here, the antifungal activities of selected fungi with high virulence against green peach aphids were tested to explore their potential for the dual control of B. cinerea and M. persicae. Antifungal activities against B. cinerea were evaluated by dual culture assays using both aerial conidia and cultural filtrates of entomopathogenic fungi. Two fungal isolates, Beauveria bassiana SD15 and Metarhizium anisopliae SD3, were identified as having both virulence against aphids and antifungal activity. The virulence of these isolates against aphids was further tested using cultural filtrates, blastospores, and aerial conidia. The most virulence was observed in the simultaneous treatment with blastospores and cultural filtrate. These results suggest that the two fungal isolates selected in this study could be used effectively for the dual control of green peach aphids and gray mold for crop protection.
Aphids* ; Beauveria ; Botrytis* ; Colon ; Crop Protection ; Farmers ; Fruit ; Fungi* ; Insecticides ; Metarhizium ; Plants ; Prunus persica ; Rhizosphere ; Spores, Fungal ; Vegetables ; Virulence

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.
Agrobacterium ; Agrobacterium tumefaciens ; Fungi ; Insects ; Metarhizium* ; Methods ; Streptothricins ; Virulence

Using dilution plating method, 47 fungal isolates were obtained from a soil sample collected from Dokdo in the East Sea of Korea in 2013. In this study, two fungal isolates, EML-MFS30-1 and EML-DDSF4, were confirmed as undescribed species, Metarhizium guizhouense and Mortierella oligospora in Korea based on current classification system using multi loci including rDNA internal transcribed spacer, large subunit, small subunit, and beta-tubulin (BTUB) genes. Herein, detailed morphological descriptions on characters of the undescribed fungal species as well as their molecular phylogenetic status are provided with comparisons to related species.
Classification* ; DNA, Ribosomal ; Korea* ; Metarhizium ; Mortierella ; Soil ; Tubulin

The beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) is difficult to control using chemical insecticides because of the development of insecticide resistance. Several pest control agents are used to control the beet armyworm. Entomopathogenic fungi are one of the candidates for eco-friendly pest control instead of chemical control agents. In this study, among various entomopathogenic fungal strains isolated from soil two isolates were selected as high virulence pathogens against larva of beet armyworm. Control efficacy of fungal conidia was influenced by conidia concentration, temperature, and relative humidity (RH). The isolates Metarhizium anisopliae FT83 showed 100% cumulative mortality against second instar larvae of S. exigua 3 days after treatment at 1 x 10(7) conidia/mL and Paecilomyces fumosoroseus FG340 caused 100% mortality 6 days after treatment at 1 x 10(4) conidia/mL. Both M. anisopliae FT83 and P. fumosoroseus FG340 effectively controlled the moth at 20~30degrees C. M. anisopliae FT83 was significantly affected mortality by RH: mortality was 86.7% at 85% RH and 13.4% at 45% RH. P. fumosoroseus FG340 showed high mortality as 90% at 45% RH and 100% at 75% RH 6 days after conidia treatments. These results suggest that P. fumosoroseus FG340 and M. anisopliae FT83 have high potential to develop as a biocontrol agent against the beet armyworm.
Beta vulgaris ; Fungi* ; Humidity ; Insecticide Resistance ; Insecticides ; Larva ; Metarhizium* ; Mortality ; Moths ; Paecilomyces* ; Pest Control ; Soil ; Spodoptera* ; Spores, Fungal ; Virulence*

OBJECTIVEThe aim of the present study was to screen the Metarhizium strains with high virulence against the larvae of Dorysthenes hydropicus, a serious pest of Citrus grandis.

METHODThirty six strains of Metarhiziums were isolated from the soil of C. grandis GAP base and collected from other institutions, and the pathogenicity of these strains against 1st instar larvae of D. hydropicus was detected at concentration of 1 x 10(8) conidia/g. The high violence strains against D. hydropicus were cultivated in sabouraud dextrose yeast medium at first, then transfer to rice grain. And the sporulations of these violent strains against D. hydropicus were detected.

RESULTTwenty-eight strains showed virulence against D. hydropicus by preliminary study, and 7 strains of them were collected for further study, 6 of the 7 showed high virulence, the highest cadaver rate was higher than 74%. The conidia production of strain 1 and strain 4 were 2.35 +/- 0. 25 (1 x 10(9) conidia/g), 2.21 +/- 0.27 (1 x 10(9) conidia/g), respectively, showed significantly higher than other strains.

CONCLUSIONStrain 1 and strain 4 of the 36 Metarhiziums strains showed high virulence against D. hydropicus, and the highest sporulation ability, so they have a best application prospect.

Animals ; Citrus ; parasitology ; Coleoptera ; microbiology ; Metarhizium ; growth & development ; isolation & purification ; pathogenicity ; Pest Control, Biological ; Plant Diseases ; parasitology ; prevention & control ; Soil Microbiology ; Spores, Fungal ; growth & development ; isolation & purification ; pathogenicity ; Virulence

A type of entomopathogenic fungus of soil in Citrus grandis 'tomentosa' production base was isolated and identified with morphological and molecular biological methods, including pathogenesis, spore characteristic and ITS sequence analysis were conducted. The results showed that eighteen entomopathogenic fungi strains were isolated from the Tenebrio molitor infected in the soil samples, which were identified as Metarhizium anisopliae var. anisopliae. Based on results above, we concluded that there was quantity of Metarhizium resources in this area. These provided the useful information for controlling some pests of C. grandis by using these strains of fungus.
Animals ; Citrus ; parasitology ; Metarhizium ; isolation & purification ; physiology ; Pest Control, Biological ; methods ; Soil Microbiology

Phosphatases play a key role not only in cell physiological functions of an organism, but also in host-pathogen interactions. Many studies demonstrated that some Gram-negative pathogenic bacteria could evade host immunity and promote pathogenicity by injecting phosphatases into host cells through type III secretion system. However, there were few reports about pathogenic fungi evading the immunity of hosts. Our researches indicated that the entomogenic fungus Metarhizium anisopliae could dephosphorylate the signal transduction substance of locust humoral immunity specifically in vitro by secreting extracellular protein tyrosine phosphatase, which implied that the fungus might interfere with the immune defense of locust. To provide reference for further studies of the functions of phosphatases, we reviewed the types of phosphatases and their roles in pathogen infection.
Animals ; Fungal Proteins ; metabolism ; Fungi ; enzymology ; physiology ; Gram-Negative Bacteria ; enzymology ; physiology ; Grasshoppers ; immunology ; microbiology ; Host-Pathogen Interactions ; Metarhizium ; enzymology ; Phosphoric Monoester Hydrolases ; classification ; physiology ; secretion