Pathogenicity and induced resistance in Larix kaempferi and Larix olgensis inoculated with Endoconidiophora fujiensis.

With climate warming and economic globalization, insect-microbe assemblages are becoming increasingly responsible for various devastating forest diseases worldwide. Japanese larch (Larix kaempferi) is extensively cultivated in China because of its high survival rate, rapid maturation, and robust mechanical properties. Endoconidiophora fujiensis, an ophiostomatoid fungus associated with Ips subelongatus, has been identified as a lethal pathogen of L. kaempferi in Japan. However, there is a dearth of research on the pathogenicity of E. fujiensis in larches in China. Therefore, we investigated the pathogenicity of E. fujiensis in introduced L. kaempferi and indigenous larch (Larix olgensis) trees and compared the induced resistance responses to the pathogen in both tree species in terms of physiology and gene expression. Five-year-old saplings and 25-year-old adult trees of L. olgensis and L. kaempferi were inoculated in parallel during the same growing season. E. fujiensis exhibited high pathogenicity in both larch species, but particularly in L. kaempferi compared to L. olgensis adult trees; adult L. olgensis was more resistant to E. fujiensis than adult L. kaempferi, which was reflected in higher accumulation of defensive monoterpenes, such as myrcene, 3-carene, and limonene, and the earlier induction of defense genes catalase (CAT) and pathogenesis-related protein 1 (PR1). This study contributes to our understanding of the interactions between bark beetle-associated ophiostomatoid fungi and host larches, from phenotypic responses to alterations in secondary metabolites via defense- and metabolism-related gene activation, providing a valuable foundation for the management of larch diseases and pests in the future.

Calcium-binding protein OsANN1 regulates rice blast disease resistance by inactivating jasmonic acid signaling.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating diseases in rice (Oryza sativa L.). Plant annexins are calcium- and lipid-binding proteins that have multiple functions; however, the biological roles of annexins in plant disease resistance remain unknown. Here, we report a rice annexin gene, OsANN1 (Rice annexin 1), that was induced by M. oryzae infection and negatively regulated blast disease resistance in rice. By yeast 2-hybrid screening, we found that OsANN1 interacted with a cytochrome P450 monooxygenase, HAN1 ("HAN" termed "chilling" in Chinese), which has been reported to catalyze the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile. Pathogen inoculation assays revealed that HAN1 was also a negative regulator in rice blast resistance. Genetic evidence showed that OsANN1 acts upstream of HAN1. OsANN1 stabilizes HAN1 in planta, resulting in the inactivation of the endogenous biologically active JA-Ile. Taken together, our study unravels a mechanism where an OsANN1-HAN1 module impairs blast disease resistance via inactivating biologically active JA-Ile and JA signaling in rice.

Pathophysiology and transcriptomic analysis of Picea koraiensis inoculated by bark beetle-vectored fungus Ophiostoma bicolor.

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors. Ophiostoma bicolor is one of the dominant species of the mycobiota associated with Ips genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in Picea koraiensis seedlings after artificial inoculation with O. bicolor strains (TS, BH, QH, MX, and LWQ). This study showed that O. bicolor was a weakly virulent pathogen of spruce, and that the virulent of the five O. bicolor strains showed differentiation. All O. bicolor strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant O. bicolor possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as Endoconidiophora spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of great significance. Traditional molecular diagnostic methods are time-consuming and require sophisticated instruments or skilled operators, which are unavailable in resource-limited settings. A specific, sensitive, and field-applicable diagnostic method is urgently needed. In this study, we developed a diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus. The false-positive signals from primer-dependent artifacts were eliminated using a probe, and base substitutions were included in the primer and probe. The entire detection process for the RPA-LFD assay can be completed under 38°C within approximately 30 min, including 15 min for crude nematode genomic DNA (gDNA) extraction and master mix preparation, 15 min for the RPA-LFD assay. This assay displayed high specificity toward B. xylophilus and showed no cross-reactions with closely related species, including Bursaphelenchus mucronatus and Bursaphelenchus doui. The sensitivity of this assay had a detection limit as low as 1 pg of B. xylophilus purified genomic DNA. Furthermore, the application of the RPA-LFD assay in simulated spiked pinewood samples showed accurate detection results. The RPA-LFD assay in this study successfully detected B. xylophilus in less than 30 min, providing a novel alternative for the simple, sensitive, and specific detection of B. xylophilus and showed potential for B. xylophilus point-of-care testing (POCT) in resource-limited areas or in field.

Diversity of fungi associated with Monochamusalternatus larval habitats in Bursaphelenchusxylophilus-infected Pinusmassoniana and identification of two new ophiostomatalean species (Ascomycota, Ophiostomatales).

Bursaphelenchusxylophilus, a pathogenic pine wood nematode (PWN), is responsible for pine wilt disease (PWD), which has caused significant economic and ecological damage worldwide, particularly in East Asia. Multiple biological factors, such as the beetle vector Monochamus, symbiotic bacteria and associated fungi, are involved in the disease infection cycle. This study isolated and identified the fungal communities of Monochamusalternatus larval galleries and pupal chambers from different instars through field investigation, morphological observation and multi-locus DNA sequence analyses in Zhejiang Province, China. A total of 255 and 454 fungal strains were isolated from M.alternatus galleries and pupal chambers infected with PWN, from the 2nd-3rd and 4th-5th instar larvae, respectively. A total of 18 species of fungi were identified, 14 species were isolated from the 2nd-3rd instar larval galleries and six species from the galleries and pupal chambers of the 4th-5th instar larvae. Amongst them were six species belonging to four genera of ophiostomatalean fungi, including two novel species, Graphilbumxianjuensis sp. nov. and Ophiostomataizhouense sp. nov. and four known species, Ceratocystiopsisweihaiensis, Ophiostomaips, Sporothrixzhejiangensis and S.macroconidia. The findings revealed that the fungal diversity and abundance of the 2nd-3rd instar larvae differed markedly from those of the 4th-5th instar larvae. This difference could be the result of fungal succession. This study provides a thorough understanding of the fungi associated with PWD and lays the groundwork for future research.

Abundance and Diversity of Ophiostomatoid Fungi Associated With the Great Spruce Bark Beetle (Dendroctonus micans) in the Northeastern Qinghai-Tibet Plateau.

The role of several virulent tree pathogens in host death has been overlooked because of the aggressiveness of their associated bark beetles. The great spruce bark beetle (Dendroctonus micans) is a widely distributed beetle that infests coniferous plants in Eurasia; however, its associated fungi have been poorly studied. Therefore, in this study, we elucidated the diversity of ophiostomatoid fungi associated with D. micans in the northeastern Qinghai-Tibet Plateau through field investigation, laboratory isolation, and culture analyses. A total of 220 strains of ophiostomatoid fungi were isolated from adults and tunnel galleries of D. micans infesting Picea crassifolia. We identified that the isolated strains belonged to eight ophiostomatoid species, including five new species (Ophiostoma huangnanense sp. nov., Ophiostoma maixiuense sp. nov., Ophiostoma sanum sp. nov., Leptographium sanjiangyuanense sp. nov., and Leptographium zekuense sp. nov.), one undefined species (Ophiostoma sp. 1), and two known species (Ophiostoma bicolor and Endoconidiophora laricicola), using phylogenetic analysis of multigene DNA sequences and morphological characteristics. This is the first time that E. laricicola, a pioneer invader and virulent pathogen, has been reported in China. We found that E. laricicola was the dominant species, accounting for 40.91% of the total number of ophiostomatoid communities. This study enriched the knowledge of the fungal associates of D. micans and elucidated that it carried the virulent pathogen E. laricicola at a surprisingly high frequency. Our findings show increased species association between D. micans and ophiostomatoid fungi and provide a basis for understanding the occurrence of forest diseases and pests.

Insights into the Synergistic Biodegradation of Waste Papers Using a Combination of Thermostable Endoglucanase and Cellobiohydrolase from Chaetomium thermophilum.

Enzymatic hydrolysis is considered an efficient and environmental strategy for the degradation of organic waste materials. Compared to mesophilic cellulases, thermostable cellulases with considerable activity are more advantageous in waste paper hydrolysis, particularly in terms of their participation in synergistic action. In this study, the synergistic effect of two different types of thermostable Chaetomium thermophilum cellulases, the endoglucanase CTendo45 and the cellobiohydrolase CtCel6, on five common kinds of waste papers was investigated. CtCel6 significantly enhanced the bioconversion process, and CTendo45 synergistically increased the degradation, with a maximum degree of synergistic effect of 1.67 when the mass ratio of CTendo45/CtCel6 was 5:3. The synergistic degradation products of each paper material were also determined. Additionally, the activities of CTendo45 and CtCel6 were found to be insensitive to various metals at 2 mM and 10 mM ion concentrations. This study gives an initial insight into a satisfactory synergistic effect of C. thermophilum thermostable cellulases for the hydrolysis of different paper materials, which provides a potential combination of enzymes for industrial applications, including environmentally friendly waste management and cellulosic ethanol production.

Characterization of a novel thermostable GH45 endoglucanase from Chaetomium thermophilum and its biodegradation of pectin.

A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni2+ affinity chromatography, and its apparent molecular mass was estimated to be 32 kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70°C and pH 4. CTendo45 was stable at 60°C for 1 h, and residual activities of 78.9% and 65.6% were estimated after 1 h at 70°C and 80°C, respectively. Ca2+, Zn2+, Mg2+, Cu2+ and Mn2+ were found to have beneficial effects on the enzyme activity to different degrees. The specific activity of purified CTendo45 was 1.52 IU mg-1 and the Km value was 59.6 µg ml-1 with a sodium carboxymethyl cellulose substrate. Moreover, CTendo45 exhibited high hydrolysis activity towards pectin, and the hydrolysis products were mainly galacturonic acid oligosaccharides. CTendo45 is the first reported bifunctional enzyme in glycoside hydrolase family 45 from C. thermophilum that is able to hydrolyze both cellulose and pectin. The biochemical properties of this recombinant CTendo45 make it a potentially effective glycoside hydrolase for industrial applications.