Occurrence of Metarhizium spp. isolated from forest samples in South India and their potential in biological control of banana stem weevil Odoiporus longicollis Oliver

Background Thirty-six entomopathogenic fungi (EPF) were isolated from soil and insect cadaver samples, collected from different forest types, viz., wet evergreen, moist deciduous, dry deciduous and scrub type in South India. Partial sequences of two parsimony informative genes ITS and RPB1 were determined under a phylogenetic approach for assessing the genetic diversity. Results Twenty-seven RPB1 gene sequences and 34 sequences of ITS1, 5.8S and ITS2 regions belonging to 36 EPF were analysed for identification and characterization. Four species of Metarhizium viz., M. anisopliae, M. roberstii, M. majus and M. guizhouense were differentiated. The isolates could be grouped into four main clades of 1–5. Most of the fungi appeared to be closely related to M. anisopliae. Based on the colony characters, colour, conidial size and shape, 27 isolates were morphologically identified as M. anisopliae. Seven strains were apparently related to M. robertsii, three isolates were similar to M. majus and the remaining one was identified as M. guizhouense. Morphological studies in congruence with phylogenetic analysis resolved the species diversity. Bioassay studies showed that M. quizhouense, M. majus and M. robertsii were effective against the banana stem weevil Odoiporus longicollis. Conclusions This is the first attempt to study the diversity and occurrence of Metarhizium species in forests of South India. Wet evergreen forest of Aralam in South India was rich in EPF diversity particularly for three species namely, M. quizhouense, M. robertsii and M. anisopliae.

Halonatospora gen. nov. with H. pansihalos comb. nov. and Glomus bareae sp. nov. (Glomeromycota; Glomeraceae)

We established single-species pot cultures of the former Glomus pansihalos, a member of the Glomeraceae, and obtained sequences of the SSU–ITS–LSU nrDNA segment and the RPB1 gene of the species. Phylogenetic analyses of the sequences indicated that G. pansihalos represents a separate clade at the rank of genus in the Glomeraceae. Consequently, the new genus was named Halonatospora, and G. pansihalos was renamed Halonatospora pansihalos comb. nov. We also grew an AMF that produced clusters with glomoid spores in single-species cultures and obtained SSU–ITS–LSU and RPB1 sequences of the fungus. Studies of pot cultures, morphological and histochemical characters of the spores, as well as phylogenetic analyses of the sequences proved that it is an undescribed species of the genus Glomus sensu stricto, which is associated with roots of Ammophila arenaria colonizing maritime sand dunes located in north-western Poland.

Hexokinase as a versatile molecular genetic marker for Microsporidia

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3′-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

Dominikia litorea, a new species in the Glomeromycotina, and biogeographic distribution of Dominikia

An arbuscular mycorrhizal fungus (AMF) producing clusters with colourless, small (11‒35 µm diam when globose) spores of unique morphological characters of two spore wall layers was grown in a trap culture and in single-species cultures. Both the spore wall layers are permanent and have the same thickness. The features of the spores prompted that the fungus most probably belongs to one of the genera, Dominikia or Kamienskia. Phylogenetic analyses of sequences of the SSU‒ITS‒LSU nrDNA and the RPB1 gene showed that the discussed AMF is an undescribed Dominikia sp. highly diverged molecularly from the 12 so far described species of the genus. Consequently, the fungus is described here as D. litorea sp. nov. The sporulation of D. litorea in the trap culture indicated that in the field the new species lived in mycorrhizal symbiosis with Xanthium spinosum that had colonized sand dunes of the Mediterranean Sea located near Verico, Greece. However, comparisons of the SSU‒ITS‒LSU sequences of D. litorea with those obtained from molecular environmental studies, which are deposited in public databases, indicated that the new species probably is also associated with roots of an unnamed plant species growing in China. In addition, based on available literature, sequence data and personal observations, the so far known geographical distribution, habitats, and plant-hosts of the described Dominikia spp. were presented and discussed. Finally, the potential participation of Dominikia spp. in influencing plants and plant communities with which they are associated and ecosystems in which they exist were discussed.

Dominikia emiratia and Rhizoglomus dunense, two new species in the Glomeromycota

The morphological, histochemical, and molecular properties of two new species of arbuscular mycorrhizal fungi (AMF; Glomeromycota) have been characterized. The first species is distinguished by spores that are orange to brownish orange, small, and formed only in clusters and mainly by having two laminate layers in a three-layered spore wall, with layer three staining dark in Melzer’s reagent. Despite the morphological similarity to some Septoglomus spp., phylogenetic analyses of sequences of the SSU–ITS–LSU nrDNA region and the RPB1 gene accommodated the fungus in the genus Dominikia, hence it was named Dominika emiratia. Intact spores of the second species, named Rhizoglomus dunense, closely resemble colourless isolates of R. clarum, but their spore wall layer three never becomes coloured with age, as does that in most R. clarum spores, and most importantly, the two fungi are separated by a large molecular distance. Dominikia emiratia was originally extracted from the rhizosphere of three plant species cultivated in two fields in a sandy desert in the Emirate of Abu Dhabi of the United Arab Emirates. Rhizoglomus dunense was found in a trap culture inoculated with the rhizosphere soil and root fragments of Ammophila arenaria, which had colonized sand dunes of the Mediterranean Sea, located near Thessalonica, Greece.

Genetic Diversity and Gene Flow of Four South African Venturia inaequalis (Apple Scab) Populations

Venturia inaequalis isolates were collected during the 2012/13 and 2013/14 seasons from the four principal apple growing regions of South Africa, Elgin (n = 114), Koue Bokkeveld (n = 126), Lower Langkloof (n = 92), and Upper Langkloof (n = 103). Sequence analysis of the ribosomal internal transcribed spacer (ITS) gene regions and genotyping with six (2012/13) and seven (2013/14) microsatellite (SSR) markers was conducted. A subset of 12 isolates from the individual ITS haplotype groups were sequenced for the translation elongation factor-1 alpha (TEF1) and the large subunit of the RNA polymerases II (RPB1) gene regions. Four haplotypes were found for ITS, whereas all isolates were identical for the TEF1 and RPB1 gene regions. The SSR markers revealed considerable variation with an average gene diversity (H) of 0.675. Multivariate analysis (discriminant analysis of principal components [DAPC]) revealed that the two Langkloof populations clustered together with the Koue Bokkeveld population. The population from the warmer winter region, Elgin, clustered separately from the rest of the populations (ΦPT = 0.076 to 0.116; P ≤ 0.05). Estimates of gene flow showed the highest migration rate from the Koue Bokkeveld, toward the Lower Langkloof (M = 151.1), and the least migration to and from the Elgin region (average M = 42.75). Occasionally, identical genotypes (clones) were detected across seasons in the Koue Bokkeveld and Elgin area, which might contribute to overwintering conidia. From this study, it is evident that South Africa most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the coastal Elgin region compared with the mountainous inland regions of the Koue Bokkeveld and the Langkloof.

Dominikia lithuanica and Kamienskia divaricata: new species in the Glomeromycota

New species in the genera Dominikia and Kamienskia (Glomeromycota) are characterized based on morphology and sequences of SSU–ITS–LSU nrDNA and the RPB1 gene. Both species produce glomoid spores only in clusters. Spores of Dominikia lithuanica are hyaline to pale yellow and 22–52 μm in diameter when globose. In their three-layered spore wall, layers 1 and 2 are almost equal in thickness and much thinner than the structural laminate layer 3. Spore wall layers 1 and 3 usually stain faintly in Melzer’s reagent. Spores of Kamienskia divaricata remain hyaline regardless of age, are 10–24 μm in diameter and have a spore wall with two layers of nearly the same thickness. The laminate layer 2 usually shows a faint dextrinoid reaction in Melzer’s reagent. A further conspicuous character of K. divaricata spores is a relatively wide subtending hypha at the spore base. In the field, D. lithuanica and K. divaricata have so far been found only twice and once, respectively, and only in maritime sand dunes of the Curonian Spit, Lithuania (D. lithuanica), and South Africa (K. divaricata). Sequence data available in public databases suggest that D. lithuanica has not yet been detected by other researchers, and K. divaricata also occurs in Texas, USA.

Multigene phylogeny and morphology reveal a new species, Ophiocordyceps tettigonia, from Guizhou Province, China

A species of Cordyceps sensu lato associated with an adult of Tettigonia was collected in Chishui Danxia natural world heritage site, Guizhou Province, China. Following morpho-phylogenetic studies it was found to be a new species and is described as Ophiocordyceps tettigonia sp. nov. It differs from similar Ophiocordyceps species in having a unique host insect, and wide secondary ascospores. Combined sequence data from the ITS, SSU, TEF, and RPB1 gene loci also confirmed the distinctiveness of this new species.

Metacordyceps shibinensis sp. nov. from larvae of Lepidoptera in Guizhou Province, southwest China

A new entomogenous taxon, Metacordyceps shibinensis sp. nov., associated with a larva of Lepidoptera was found in Yuntai Mountains, Guizhou Province, China. It differs from similar species in its white to faint yellow stromata, short ascomata, and very short asci and ascospores. Combined sequence analyses of 5.8S-ITS rDNA, nrSSU, EF-1α and RPB1 gene-loci also confirmed the distinctiveness of this new species.

Systematic analyses of Ophiocordyceps ramosissimum sp. nov., a new species from a larvae of Hepialidae in China

A new species, Ophiocordyceps ramosissimumsp. nov., is described and illustrated. It was associated with larvae of Phassus nodus (Hepialidae) collected from Xuefeng Mountains, Hunan Province, China. It differs from similar species in having branched stromata without a sterile apex, superficial ascomata, and very wide asci and ascospores and in its occurrence on Phassus nodus in living roots or trunks of Clerodendrum cyrtophyllum. Multi-gene phylogenetic analysis of 5.8S-ITS rDNA, nrSSU, EF-1α, and RPB1 gene loci also confirmed the distinctiveness of this new species.