Majus Isolates Research Articles

Virulence of Metarhizium isolated from infected Oryctes rhinoceros L. (Coleoptera: Scarabaeidae) larvae around coconut plantations in East Java, Indonesia

BackgroundMetarhizium is one of the entomopathogenic fungi (EPF) that has been widely reported as a useful agent for controlling the coconut rhinoceros beetle (CRB), Oryctes rhinoceros L. (Coleoptera: Scarabaeidae). Application of this fungus as a biopesticide is influenced by regional environmental conditions that affect the pathogenic activity against the targeted pest. Several studies have proven that the native fungal isolates have strong pathogenic activity than EPF introduced from other regions. The identification of local EPF species is a strategic approach to develop potential biopesticides with standard properties including host specificity, climate suitability, and significantly suppressing the target pest population. This study aimed to identify Metarhizium species isolated from infected CRB larvae with high pathogenicity to host pests in East Java, Indonesia.ResultsThirteen isolates were obtained which were divided into 4 clades based on phylogenetic analysis by the ITS rDNA region, namely M. anisopliae var. lepidiotae, M. anisopliae var. anisopliae, M. brunneum, and M. majus. Identified Metarhizium species exhibited varied sizes of conidia, but fell within the size ranges reported in previous studies. Interestingly, the isolate MaSi produced conidial lengths well above the range of conidial sizes recorded and placed these isolates (M. anisopliae) in the MGT clade, known as MALC (M. anisopliae s.l. with large conidia). The present investigation reported that isolates MaLe, MaMa, MaWa, and MaSi produced higher mortality values than other isolates, supporting that M. majus and MALC had higher pathogenicity against O. rhinoceros larvae than other Metarhizium species. Neighbor-joining analysis showed a close resemblance between the isolate MaMa and the strain ARSEF 1946, which was isolated from the CRB. In addition, the isolate MaMa had the highest virulence against O. rhinoceros larval cadaver with a faster lethal time (for 50% mortality). This result indicated a possible relationship between phylogenetic status or DNA sequence polymorphisms with Metarhizium pathogenicity and host specificity.ConclusionsThe local species of Metarhizium have been isolated from infected CRB larvae, and M. majus isolates exhibited high pathogenicity against O. rhinoceros larvae. The close similarity of M. majus isolates and CRB-isolated strains suggests a possible relationship between pathogenicity and host specificity with phylogenetic status or DNA sequence polymorphisms.

First Report of Microdochium nivale and M. majus Causing Brown Foot Rot of Wheat in China.

Microdochium nivale and M. majus not only cause seedling blight of wheat (Triticum aestivum L.) in cold dry soils, but also cause foot rot and ear blight of wheat under favorable conditions (Haigh et al. 2009). In May 2017, 2019, and 2020, a serious foot rot of wheat with an incidence of 92%, 45%, and 51% was observed in the field in Xiangcheng County (33.43° N, 114.84° E), Tanghe County (32.43° N, 112.66° E), and Linzhou City (36.13° N, 113.75° E), Henan Province, respectively. The serious brown lesions of the lower leaf sheaths is visible. The pathogens were isolated from brown leaf sheaths on potato dextrose agar (PDA) after being surface-sterilized (70% EtOH for 30 s followed by 3% NaClO for 1.5 min) and rinsed three times in sterile distilled water. After 5 d, mycelia were transferred to fresh PDA, and nine representative isolates (G17ZK2-1, G17ZK2-2, G17ZK2-3, g19TH10-4, g19TH10-5, g19TH10-6, G20LZ1-6, G20LZ1-7, and G20LZ1-8) were further purified by hyphal tipping. Species were identified based on morphological characteristics, and sequence analysis of partial sequences of the translation elongation factor-1α (TEF), the RNA polymerase II subunit (RPB2) gene and β-tubulin gene (Abdelhalim et al. 2020). Among the nine isolates, six isolates belonged to M. majus, three isolates belonged to M. nivale. Sequences of six isolates M. majus and three isolates M. nivale were deposited in GenBank with accession numbers MW428296-MW428298, MZ734119-MZ734121and MZ734139-MZ734141(TEF), MW384889, MW428291, MW428292, MZ734203-MZ734205 and MZ734161-MZ734163(RPB2), MW428293-MW428295, MZ501004-MZ501006 and MZ501024-MZ501026 (β-tubulin). For all the genes, isolates revealed 98-100% similarity to M. majus and M. nivale accessions, respectively. Microscopy of the six M. majus isolates showed: the conidia were falcate, straight to curved, apex pointed or obtuse to subacute, lacking basal differentiation, with 1 to 6 septa, 3.6 to 5.0 × 15.0 to 30.5 μm (av.= 4.5 × 23.2; n = 60). The three M. nivale isolates showed: the conidia were hyaline, 1 to 3 septa, 2.4 to 4.4 × 11.9 to 26.0 μm (av.= 3.5 × 14.7; n = 60). Perithecia of M. majus are dark brown, globose, and 95.2 to 190.5 × 95.2 to 228.6 μm (av.= 144.4 ×152.5; n = 30). Asci are clavate, and 6.8 to 11.0 ×68.2 to 77.3 μm (av.= 8.6×72.0; n = 30), contain eight ascospores. Mature ascospores are ellipsoidal, and 3.8 to 4.9 ×11.5 to 19.2 μm (av.= 4.0 ×15.2; n = 30), with 1 to 3 septa. These morphological characteristics were consistent with previous descriptions of these two species (Glynn et al. 2005). For pathogenicity tests, mycelia of M. nivale and M. majus was prepared using the modified procedure of Zhang et al. (2015). Two-week-old healthy wheat seedlings (cv. Aikang 58) were inoculated using 1 mL of prepared mycelia to one seedling, which was sprayied on soil. Control seedlings were inoculated with 1 mL distilled water containing 0.2% gelatin. After 10 days under 15/10℃, 16h/8h, all the inoculated plants had developed brown spots; while control plants remained healthy. The pathogens were reisolated from inoculated plants and identified as M. nivale and M. majus based on morphological characteristics and molecular methods described above. Although there are reports of M. majus associated with brown foot rot of wheat in Anhui Province and M. nivale associated with seedling blight of oat in Gansu Province (Chen et al. 2021; Tai et al. 2019). To our knowledge, this is the first report of brown foot rot of wheat caused by M. nivale and M. majus in China.

Pathogenicity, host specificity and genetic diversity in Norwegian isolates of Microdochium nivale and Microdochium majus

Microdochium majus and Microdochium nivale cause serious disease problems in grasses and cereal crops in the temperate regions. Both fungi can infect the plants during winter (causing pink snow mould) as well as under cool humid conditions during spring and fall. We conducted a pathogenicity test of 15 M. nivale isolates and two M. majus isolates from Norway at low temperature on four different grass cultivars of Lolium perenne and Festulolium hybrids. Significant differences between M. nivale isolates in the ability to cause pink snow mould were detected. The M. nivale strains originally isolated from grasses were more pathogenic than isolates from cereals. The genetic diversity of M. nivale and M. majus isolates was studied by sequencing four genetic regions; Elongation factor-1 alpha (EF-1α), β-tubulin, RNA polymerase II (RPB2) and the Internal Transcribed Spacer (ITS). Phylogenetic trees based on the sequences of these four genetic regions resolved M. nivale and M. majus isolates into separate clades. Higher genetic diversity was found among M. nivale isolates than among M. majus isolates. M. nivale isolates revealed genetic differences related to different host plants (grasses vs. cereals) and different geographic regions (Norway and UK vs. North America). Sequence results from the RPB2 and β-tubulin genes were more informative than those from ITS and EF-1α. The genetic and phenotypic differences detected between Norwegian M. nivale isolates from cereals and grasses support the assumption that host specialization exist within M. nivale isolates.

Phylogenetic status and pathogenicity of Metarhizium majus isolated from a fruit beetle larva in Japan

Some isolates of Metarhizium anisopliae sensu lato with large conidia (MALC), which was formerly known as M. anisopliae var. majus, from a scarabaeid host, such as isolates from the coconut rhinoceros beetle (Oryctes spp.), have a narrow host range and are particularly adapted to their original host. Despite recent taxonomic revisions of the genus Metarhizium on the basis of DNA sequences, variations in host preferences of MALC have not been linked to DNA sequence variations. This study focused on the phylogenetic status and pathogenicity of MALC isolated from the fruit beetle larva Protaetia orientalis submarumorea (Pos) in Japan to investigate the relationship between virulence and nucleotide sequence variation among MALC. On the basis of the results of the phylogenetic analyses of DNA sequences from five loci, we identified an isolate from Pos (Hn1) as M. majus, which comprised many isolates with large conidia from scarabaeid insects. In the virulence assay of genetically diverse Metarhizium isolates, only Hn1 and its most closely related isolates from soil showed pathogenicity to Pos; however, these Pos-pathogenic isolates showed weak virulence against silkworms. In the analysis of the intergenic spacer region of rDNA, the Pos-pathogenic isolates displayed unique sequence variation and were clearly distinguished from closely related lineages, including isolates from Oryctes sp., which formed a separate monophyletic group. These results indicate that the Pos-pathogenic isolates from Japan are particularly adapted to Pos and can be genetically distinguished from M. majus isolates from different scarabaeid insects.

Multigene differences betweenMicrodochium nivaleandMicrodochium majus

Microdochium nivale (Fr.) Samuels & Hallett and Microdochium majus (Wollenw.) Glynn & S.G. Edwards are sister species that cause diseases on grasses and cereals at low temperatures. The DNA sequences of RPB2 (RNA polymerase II), β-tubulin, EF-1α (elongation factor), and ITS (rDNA internal transcribed spacer) from these groups were analysed to compare the extent of differences between these species, among isolates from Europe compared with those from North America, and among isolates of M. nivale originally collected from Agrostis spp. compared with isolates from wheat (Triticum aestivum). All of the regions studied except for ITS resolved M. nivale and M. majus isolates into separate clades. The RPB2 sequences also resolved both the North American and European M. majus isolates and M. nivale isolates from either turfgrasses or wheat into separate clades. These results support the recent elevation of M. nivale and M.majus to sister species and also provide some support for the assertion that there may be host-specific differences among M. nivale, which has a wider host range than M. majus.

Specific PCR Detection of Four Quarantine Fusarium Species in Korea

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

First occurrence of resistance to strobilurin fungicides in Microdochium nivale and Microdochium majus from French naturally infected wheat grains

Microdochium nivale (Fr.) Samuels & Hallet and Microdochium majus (Wollenweber) belong to the Fusarium ear blight (FEB) fungal complex affecting cereals. In 2007 and 2008, major Microdochium sp. infestations were observed in France, and the efficacy of strobilurins was found to be altered in some field trials. The aim of this study was to determine the sensitivity to strobilurins of French isolates of Microdochium and to characterise the possible mechanisms of resistance. Half of the strains collected in 2007 were resistant to strobilurins, and most also displayed strong resistance to benzimidazoles. Strobilurin resistance was found mostly in M. majus isolates. Positive cross-resistance was observed between all strobilurins tested, but not with the phenylpyrrole derivative fludioxonil and the various classes of sterol biosynthesis inhibitors (SBIs). In most strains, resistance was correlated with the G143A substitution in cytochrome b, the molecular target of strobilurins. Two other mechanisms were also detected at lower frequencies. This is the first report of strobilurin resistance in Microdochium. Several resistance mechanisms have evolved independently in populations and may have different impacts on field efficacy. This makes the accurate detection and quantification of QoI resistance difficult. The management of field resistance and efficacy must be adapted to take these findings into account.

Isolates of Microdochium nivale and M. majus Differentiated by Pathogenicity on Perennial Ryegrass (Lolium perenne L.) and in vitro Growth at Low Temperature

AbstractPink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β‐glucosidase, a cell wall‐degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence‐based evidence for the elevation of these former varieties to species status.

Towards the Development of a Novel In Vitro Strategy for Early Screening of Fusarium Ear Blight Resistance in Adult Winter Wheat Plants

A novel in vitro bioassay is described for screening Fusarium ear blight (FEB) resistance in adult winter wheat plants. Seven winter wheat cultivars were assessed for components of partial disease resistance as 28 day-old detached leaf segments in the laboratory using isolates of Microdochium nivale var. nivale and M. nivale var. majus. Results were compared with disease data obtained at anthesis using the same cultivars as whole plants and the same isolates under glasshouse conditions. Significant cultivar differences were observed using detached leaves, with cv. Avalon (a Fusarium culmorum ear susceptible cultivar) having the shortest leaf incubation period, greatest leaf lesion development and shortest leaf latent period compared to cv. Spark (a Fusarium culmorum ear resistant cultivar), which had the longest leaf incubation period, least leaf lesion development and longest leaf latent period. Using whole plants, cv. Avalon had the shortest ear incubation period and greatest ear disease severity, whilst cv. Spark had the longest incubation period and least ear disease severity. Overall, cultivars of intermediate F. culmorum ear resistance expressed intermediate responses to M. nivale isolates, using both detached leaves and whole plants. Significant correlations were found with ear disease severity and ear incubation period in whole plants and components of partial disease resistance in detached leaves, with significant correlations obtained between leaf incubation period and ear disease parameters using the M. nivale var. nivale isolate. In addition, leaf latent period and leaf lesion size showed significant correlations with whole plant reactions using M. nivale var. nivale and var. majus isolates. The in vitro screening of cultivars as detached leaves using M. nivale isolates may offer a real possibility of a rapid bioassay for the early screening of FEB resistance in wheat and other cereals.

Analysis of Microdochium nivale isolates from wheat in the UK during 1993

SUMMARYA total of 144 isolates of Microdochium nivale from stem bases of winter wheat were taken from 30 sites and 91 isolates from grain were taken from seven sites throughout the UK. Identification by polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region followed by restriction enzyme digestion of the PCR product revealed that 70% of stem base isolates were M. nivale var. majus and 30% var. nivale while 93% grain isolates were var. majus and 7% var. nivale. Almost all isolates were resistant to the benzimidazole fungicide benomyl. Perithecial production in vitro was more common in var. majus isolates and occurred in almost all grain isolates, but was less common in stem base isolates. The implications of the findings in terms of epidemiology and chemical control of this important cereal pathogen are discussed.

Restriction fragment length polymorphisms in the ribosomal RNA gene complex of isolates of the entomopathogenic fungus Metarhizium anisopliae

Restriction fragment length polymorphisms (RFLPs) in DNA coding for ribosomal RNA (rDNA) were analysed from different species and isolates of Metarhizium. M. anisopliae var. anisopliae, M. anisopliae var. majus, M. album and M. flavoviride could be differentiated by the presence of restriction enzyme sites in the rDNA repeat unit. Isolates of M. anisopliae could be differentiated according to their geographical origin after cluster analysis of rDNA RFLPs, but there was no apparent correlation between RFLPs and original insect host. Some M. anisopliae var. majus isolates were shown to be heterozygous diploids.

Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae

In this study, we examined variation among isolates of the entomopathogenic fungus Metarhizium anisopliae in their ability to germinate and produce infection structures (appressoria) in different nutrient media. We also examined allozyme variation within and among 120 isolates of Metarhizium spp. Electromorph differences were noted and genotypic designations were assigned for eight loci encoding the following enzymes: glucose phosphate isomerase, glutamate dehydrogenase, glutamic pyruvic transaminase, glutathione reductase, malate dehydrogenase, phosphogluconate dehydrogenase, peptidase with glycyl leucine, and triosephosphate isomerase. Forty-eight distinct genotypic classes were found. Only the thirteen M. anisopliae var. majus isolates and an isolate of Metarhizium flavoviride possessed multibanded phenotypes characteristic of heterozygous genotypes. Three isolates were heterozygous at one locus, seven isolates at two loci, and four isolates at four loci. Based on the level of genetic similarities between the three recognized species ( M. anisopliae, Metarhizium album, and M. flavoviriae) we report that isolates currently assigned as M. anisopliae are a strongly heterogenous array of multilocus genotypes that includes five varieties and two undescribed cryptic species. Genetic similarities provided evidence for geographical clustering. For example, Brazil and Colombia contain a common, widely dispersed genotypic class; eight additional classes are more locally distributed. Nutrient requirements for germination and appessorium formation were frequently host related, irrespective of genetic and geographical distance. Most of those lines of M. anisopliae var. anisopliae and var. majus isolated from scarabaeids germinated readily only in the presence of a crude protein/chitin product. Lines isolated from other coleopterans were much more adaptable and produced appressoria in yeast extract media. Unlike the majority of isolates from other hosts, lines isolated from Hemiptera usually germinated well in media containing glucose as the sole carbon source, and glucose allowed formation of appressoria. The role of selectable strain variations in the development of pathogen strategies is discussed.

Use of the optical brightener Tinopal BOPT for the rapid determination of conidial viabilities in entomogenous deuteromycetes

Viable conidia of Beauveria bassiana and Metarhizium anisopliae fluoresced only weakly after staining in bi-striazinyl amino stilbene (0·05% Tinopal BOPT) and exposure to uv light. By contrast, non-viable conidia fluoresced brightly and could be distinguished from viable spores. A comparison between the fluorescent method and a conventional technique, measuring conidial germination on agar gave good correlations between the two methods for all five B. bassiana isolates examined. With M. anisopliae the two methods gave similar results for var. anisopliae strains but not for var. majus isolates. The fluorescent method was used to determine viabilities of lyophilized B. bassiana conidia stored at 5 and 20 °C. Good correlation between the two methods was obtained up to 451 d at the lower temperature but only for 95 d at 20°. The implications of the results are discussed.

Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens

Laboratory bioassays were conducted to assess the pathogenicity of 24 wild-type isolates of Metarhizium anisopliae toward Nilaparvata lugens. LT 50 values ranged from 5 days (highly pathogenic) to > 14 days (nonpathogenic). In vitro germination, growth rate, spore production, extracellular enzyme production, and spore characteristics were assessed in relation to pathogenicity. Rapid germination and growth, and small spore volume, were correlated with high pathogenicity. Highly pathogenic isolates produced no detectable extracellular amylase and detectable amounts of extracellular chitinase, lipase, and protease activity. Evidence for the stable diploid nature of var. majus isolates is presented.