Zinc solubilization and organic acid production by the entomopathogenic fungus, Metarhizium pingshaense sheds light on its key ecological role in the environment.

We report for the first-time higher zinc (Zn) solubilization efficiency and plant growth promotion by an entomopathogenic fungus (EPF), Metarhizium pingshaense IISR-EPF-14, which was earlier isolated from Conogethes punctiferalis, a pest of global importance. The Zn solubilizing efficiency of the fungus varied depending on the type of insoluble source of Zn used, which was observed to be 1.6 times higher in Zn3(PO4)2-amended media compared to ZnO media. In liquid media, there was a 6.2-fold increase in available Zn in ZnO-amended media, whereas a 20.2-fold increase in available Zn was recorded in Zn3(PO4)2 medium. We ascribe the production of various organic acids such as gluconic, keto-gluconic, oxalic, tartaric, malonic, succinic and formic acids, which in general, interact with insoluble Zn sources and make them soluble by forming metal cations and displacing anions as the major mechanism for Zn solubilization by M. pingshaense. However, the type and amount of organic acid produced in the media varied depending on the source of Zn used and the incubation period. Application of the fungus alone and in combination with insoluble Zn sources enhanced various plant growth parameters in rice and cardamom plants. Moreover, the uptake of Zn in rice plants was enhanced up to ~2.5-fold by fungal application. The fungus also exhibited various other plant growth-promoting traits, such as production of Indole-3-acetic acid, ammonia, siderophores, solubilization of mineral phosphate, and production of hydrolytic enzymes such as α-amylase, protease, and pectinase. Hence, apart from its use as a biological control agent, M. pingshaense has the potential to be used as a bio-fortifier to enhance the solubilization and uptake of Zn from nutrient poor soils under field conditions. Our findings shed light on the broader ecological role played by this fungus and widen its scope for utilization in sustainable agriculture.

Metarhizium indicum, a new species of entomopathogenic fungus infecting leafhopper, Busoniomimus manjunathi from India.

A new species of entomopathogenic fungus, Metarhizium indicum, which derives its species epithet after its Indian origin is reported here. The fungus was found to cause natural epizootics in leafhopper (Busoniomimus manjunathi) infesting Garcinia gummi-gutta (Malabar tamarind), an evergreen spice tree native to South and Southeast Asia, known for its use as a culinary flavourant, dietary supplement and traditional remedy for various human ailments. The fungus was found to cause more than 60% mortality in field collected insects. The identity of the new species was established based on its distinct morphological characteristics and multi-gene sequence data analyses. Phylogenetic analyses using internal transcribed spacer region (ITS), DNA lyase (APN2) and a concatenated set of four marker genes [translation elongation factor 1-alpha (TEF), ß-tubulin (BTUB), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2)] along with marked differences in nucleotide composition and genetic distance unambiguously support our claim that the present fungus infecting Garcinia leafhopper is a new addition to the genus Metarhizium.

Characterization and biocontrol potential of a naturally occurring isolate of Metarhizium pingshaense infecting Conogethes punctiferalis.

An entomopathogenic fungus was isolated from an infected larva of Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), a highly polyphagous pest recorded from more than 120 plants and widely distributed in Asia and Oceanic countries. The fungus was identified as Metarhizium pingshaense Q.T. Chen & H.L. Guo (Ascomycota: Hypocreales) based on morphological characteristics and molecular studies. Scanning electron microscopic studies were conducted to study the infection of C. punctiferalis by M. pingshaense. Bioassay studies with purified conidial suspension proved that the isolate was highly virulent to C. punctiferalis, causing more than 86 % mortality to fifth instar larvae at 1 × 108 spores/mL, under laboratory conditions. The median lethal concentration (LC50) of the fungus against late instar larvae was 9.1 × 105 conidia/mL and the median survival time (MST) of late instar larvae tested at the doses of 1 × 108 and 1 × 107 conidia/mL were 4.7 and 6.4 days, respectively. The optimal temperature for fungal growth and sporulation was found to be 25 ± 1 °C. This is the first report of M. pingshaense naturally infecting C. punctiferalis. Isolation of a highly virulent strain of this fungus holds promise towards development of a potential mycoinsecticide against this pest.

Multifarious plant growth promotion by an entomopathogenic fungus Lecanicillium psalliotae.

An entomopathogenic fungus, Lecanicillium psalliotae strain IISR-EPF-02 previously found infectious to cardamom thrips, Sciothrips cardamomi promoted plant growth in cardamom, Elettaria cardamomum. The isolate exhibited direct plant growth promoting traits by production of indole-3-acetic acid and ammonia and by solubilizing inorganic phosphate and zinc. It also showed indirect plant growth promoting traits by producing siderophores and cell wall-degrading enzymes like, α-amylases, cellulases and proteases. In pot culture experiments, application of the fungus at the root zone of cardamom seedlings significantly increased shoot and root length, shoot and root biomass, number of secondary roots and leaves and leaf chlorophyll content compared to untreated plants. This is the first report on the plant growth promoting traits of this fungus. The entomopathogenic and multifarious growth promoting traits of L. psalliotae strain IISR-EPF-02 suggest that it has great potential for exploitation in sustainable agriculture.

Characterization and virulence of Beauveria bassiana associated with auger beetle (Sinoxylon anale) infesting allspice (Pimenta dioica).

The incidence of auger beetle, Sinoxylon anale Lesne (Bostrichidae: Coleoptera), a destructive pest of cosmopolitan occurrence is reported for the first time on allspice trees, Pimenta dioica (L.) Merr. in Kerala, India. The insects bored through the basal region of fresh twigs resulting in dieback symptoms. Morphological characterization and sequencing of a partially amplified fragment of the mitochondrial CO1 gene (696bp) revealed the insect to be Sinoxylon anale. An entomopathogenic fungus was isolated from infected cadavers of S. anale that was identified as Beauveria bassiana (Bals.-Criv.) Vuill., sensu stricto (s.s.) (Ascomycota: Hypocreales) based on morphological and molecular studies. The partial sequences of the ITS, TUB, TEF and Bloc gene regions were sequenced. The fungus grew well in ambient room temperature conditions (28-32±2°C; 60-70% relative humidity) and the infection process on the insect was documented by scanning electron microscopy. Bioassay studies with the isolate indicated that the fungus was virulent against adult beetles as evidenced by the LC50 (3.6×10(6)conidia/ml) and ST50 values (6.8days at a dose of 1×10(7)conidia/ml and 5.8days at a dose of 1×10(8)conidia/ml, respectively). This is the first record of B. bassiana naturally infecting S. anale and the fungus holds promise to be developed as a mycoinsecticide.

Occurrence and characterization of a tetrahedral nucleopolyhedrovirus from Spilarctia obliqua (Walker).

Spilarctia obliqua Walker (Lepidoptera: Arctiidae) is a polyphagous insect pest damaging pulses, oil seeds, cereals, vegetables and medicinal and aromatic plants in India. The pest also infests turmeric and ginger sporadically in Kerala. We observed an epizootic caused by a nucleopolyhedrovirus (NPV) in field populations of the insects in December 2013. The NPV was purified and characterized. The isolate was tetrahedral in shape and belonged to multicapsid NPV. The REN profile of the SpobNPV genome with Pst I, Xho I and HindIII enzymes showed a genome size of 99.1±3.9 kbp. Partialpolh, lef-8 and lef-9 gene sequences of the isolate showed a close relationship with HycuNPV and SpphNPV. Phylogram and K-2-P distances between similar isolates suggested inclusion of the present SpobNPV isolate to group I NPV. The biological activity of the isolate was tested under laboratory conditions against third instar larvae of S. obliqua and the LC50 was 4.37×10(3)OBs/ml occlusion bodies (OBs) per ml. The median survival time (ST50) was 181 h at a dose of 1×10(6)OBs/ml and 167 h at a dose of 1×10(8)OBs/ml. SpobNPV merits further field evaluation as a potential biological control agent of S. obliqua, a serious pest of many agriculturally important crops in the Oriental region.