Development of a recombinase polymerase amplification method combined with a lateral flow dipstick assay for rapid detection of the larch pathogen Neofusicoccum laricinum.

Neofusicoccum laricinum, an important pathogenic species, causes shoot blight of larch. In China, large areas of Larix principis-rupprechtii forests are threatened by this pathogen. Currently, this pathogen is on the list of quarantine pests in Chinese. Due to the widespread and severe damage caused by N. laricinum, a reliable and accurate diagnostic tool is urgently needed. In this study, we first identified a Nlar12009 as a N. laricinum-specific gene through genomic sequence data and bioinformatic analysis. Specific primer pairs and DNA probes were designed to detect the target pathogen using a novel recombinase polymerase amplification assay with a lateral flow dipstick (RPA-LFD) method. We optimized the RPA-LFD assay to ensure high specificity to N. laricinum. Our results showed that the assay exclusively detected N. laricinium isolates with no cross-reaction with other isolates of fungaland oomycete species and nematodes. Furthermore, our detection technique exhibited a 10-fold higher sensitivity (10 fg/mL) than conventional polymerase chain reaction (PCR) for N. laricinum detection. Our developed RPA-LFD assay is proved to be a highly specific, sensitive, time-saving, and convenient method for the diagnosis of N. laricinum and shows great potential in field application.

Evaluation of the novel endophytic fungus Chaetomium ascotrichoides 1-24-2 from Pinus massoniana as a biocontrol agent against pine wilt disease caused by Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD), is an ever-increasing threat to Pinus forests worldwide. This study aimed to develop biological control of PWD by the application of endophytic fungi isolated from healthy pine trees. RESULTS: We successfully isolated a novel endophytic fungal strain 1-24-2 from branches of healthy Pinus massoniana. The culture filtrates (CFs) of strain 1-24-2 exhibited strong nematicidal activity against Bursaphelenchus xylophilus, with a corrected mortality rate of 99.00%. Based on the morphological and molecular characteristics, the isolated strain 1-24-2 was identified as Chaetomium ascotrichoides. In the in-planta assay, pine seedlings (2-years-old) treated with 1-24-2 CFs + pine wood nematode (T2) showed a significant control effect of 80%. A total of 24 toxic compounds were first identified from 1-24-2 CFs through gas chromatography-mass spectrometry (GC-MS) analysis, from which O-methylisourea, 2-chlorobenzothiazole, and 4,5,6-trihydroxy-7-methylphthalide showed robust binding sites at Tyr119 against phosphoethanolamine methyltransferase (PMT) protein of Bursaphelenchus xylophilus by molecular docking approach and could be used as potential compounds for developing effective nematicides. Interestingly, strain 1-24-2 produces toxic volatile organic compounds (VOCs), which disturb the natural development process of B. xylophilus, whose total number decreased by up to 83.32% in the treatment group as compared to control and also reduced Botrytis cinerea growth by up to 71.01%. CONCLUSION: Our results highlight the potential of C. ascotrichoides 1-24-2 as a promising biocontrol agent with solid nematicidal activity against B. xylophilus. This is the first report of C. ascotrichoides isolated from P. massoniana exhibiting strong biocontrol potential against B. xylophilus in the world. © 2024 Society of Chemical Industry.

Screening of bacterial endophytes of larch against Neofusicoccum laricinum and validation of their safety.

In order to provide a highly feasible research pathway for the control of larch shoot blight, healthy larch branches and leaves were collected from 13 sampling sites in 8 provinces in China. The antagonistic endophytic bacteria obtained from the screening were used to carry out disease control experiments in potted seedlings. The safety evaluation test was conducted on the antagonistic bacteria. Subsequently, the strains with better preventive effect and high safety were identified by morphological and molecular methods. A total of 391 strains of endophytic bacteria were isolated from healthy larch branches and leaves. Seventy-eight strains of larch endophytic bacteria with antagonistic effect were obtained by primary sieving. Ten strains of endophytic bacteria with obvious antagonism were further obtained by secondary sieving, and all of them had an inhibition rate of more than 57%. Among them, strains YN 2, JL 6, NMG 23, and JL 54 showed the highest inhibition rate of 63.16%-65.08%, which was significantly different from the other treatments. The results of the pot test showed that 14 days after inoculation with the pathogen, strains YN 2 and JL 54 were more effective in the control of larch shoot blight, with the preventive effects reaching 57.7% and 50.0%, respectively. Strains JL 6 and JL 54 were biologically safe in the safety evaluation test. Therefore, strain JL 54 was selected for identification. It was identified as Bacillus amyloliquefaciens through morphological observation, 16S rDNA sequence, gyrB gene sequence and 16S rDNA-gyrB tandem feature sequence analysis. IMPORTANCE: Larch shoot blight is a widely distributed, damaging, and rapidly spreading fungal disease of forest trees that poses a serious threat to larch plantations. Endophytic bacteria have biological effects on host plants against pests and diseases, and they have a growth-promoting effect on plants. In this paper, we investigated for the first time the biocontrol effect of endophytic bacteria on larch shoot blight by screening endophytic bacteria with the function of antagonizing dieback fungi. Bacillus amyloliquefaciens JL 54 has a better prospect of biocontrol against larch shoot blight, which lays the foundation for the application of this bacterium in the future.

Transcriptome analysis of Euwallacea interjectus reveals differentially expressed unigenes related to developmental stages and egg laying.

Euwallacea interjectus (Curculionidae: Scolytinae) is an ambrosia beetle species in its early stages of research. Therefore, studying the related molecular mechanism associated with the development and egg laid is essential. Transcriptome sequencing was used in this study to compare the gene expression of the beetles at different developmental stages and female adults before and after oviposition. A total of 40,047 annotated unigenes were obtained. There were 4225 differentially expressed unigenes (DEUs) from larva to prepupa stage, 3651 DEUs between prepupa and pupa, 1675 DEUs generated from pupa to adult, and 4762 DEUs between females before and after oviposition. The most significant pathway differences between different development stages and before and after oviposition were selected through functional annotation of DEUs between different stages. Among them, there were many pathways related to protein metabolism including: neuroactive ligand-receptor interaction, endoplasmic reticulum and RNA transport. This study provides valuable information on the molecular regulation mechanism of development and the egg laid of E. interjectus.

Effect of Associated Bacteria GD1 on the Low-Temperature Adaptability of Bursaphelenchus xylophilus Based on RNA-Seq and RNAi.

To explore the effect of associated bacteria on the low-temperature adaptability of pinewood nematodes (PWNs), transcriptome sequencing (RNA-seq) of PWN AH23 treated with the associated bacterial strain Bacillus cereus GD1 was carried out with reference to the whole PWN genome. Bioinformatic software was utilized to analyze the differentially expressed genes (DEGs). This study was based on the analysis of DEGs to verify the function of daf-11 by RNAi. The results showed that there were 439 DEGs between AH23 treated with GD1 and those treated with ddH2O at 10 °C. There were 207 pathways annotated in the KEGG database and 48 terms annotated in the GO database. It was found that after RNAi of daf-11, the survival rate of PWNs decreased significantly at 10 °C, and fecundity decreased significantly at 15 °C. It can be concluded that the associated bacteria GD1 can enhance the expression of genes related to PWN low-temperature adaptation and improve their adaptability to low temperatures.

An alkaline protease from Bacillus cereus NJSZ-13 can act as a pathogenicity factor in infection of pinewood nematode.

Endophytic bacteria are an important biological control for nematodes. We isolated the nematicidal Bacillus cereus NJSZ-13 from healthy Pinus elliottii trunks. Bioassay experiments showed killing of all tested nematodes by proteins from the NJSZ-13 culture filtrate within 72 h. Degradation of the nematode cuticles was observed, suggesting the action of extracellular bacterial enzymes. The responsible protease was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography, and SDS-PAGE. The protease had a molecular weight of 28 kDa and optimal activity at 55 °C and pH 9, indicating an alkaline protease. The study suggests the potential for using this B. cereus NJSZ-13 strain protease to prevent pinewood nematode infection.

Jasmonate induced terpene-based defense in Pinus massoniana depresses Monochamus alternatus adult feeding.

BACKGROUND: Symptoms of pine wilt disease (PWD) are caused when pathogenic pine wood nematodes (PWN) invade healthy host trees via wounds created by adult Monochamus alternatus. Exogenous methyl jasmonate (MeJA) can trigger terpene-based induced defense in conifers, which is a core part of the conifer defense system. Herein, we hypothesized that the terpene-based plant defense induced by MeJA could negatively affect the feeding behavior of M. alternatus adults, and may contribute to a new strategy in the control of PWD. RESULTS: The feeding area for M. alternatus adults feeding on MeJA-treated seedlings was significantly smaller compared with control seedlings. However, no significant difference was detected in the enzyme activities in the midgut of beetles that had fed on these seedlings. Terpenoids were mainly accumulated in traumatic resin duct, whereas the constitutive resin duct accumulated only diterpenoids. Correspondingly, large-scaled responses at the transcriptional level mainly focused on terpenoid and phenolic biosynthesis in the defending trees. CONCLUSIONS: Breeding tree species with a high resin yield may contribute to control of the spread of PWD by suppressing the feeding of M. alternatus adults. Transcriptome sequencing results provided abundant information for further breeding of highly resistant trees. Based on these findings, a potential push-pull strategy for the control of M. alternatus was discussed.

Characterization, expression profiling, and thermal tolerance analysis of heat shock protein 70 in pine sawyer beetle, Monochamus alternatus hope (Coleoptera: Cerambycidae).

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35-50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2-3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25-40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity.

Bacterial Community Structure of Pinus Thunbergii Naturally Infected by the Nematode Bursaphelenchus Xylophilus.

Pine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a devastating disease in conifer forests in Eurasia. However, information on the effect of PWD on the host microbial community is limited. In this study, the bacterial community structure and potential function in the needles, roots, and soil of diseased pine were studied under field conditions using Illumina MiSeq coupled with Phylogenetic Investigation of Communities by Reconstruction of Unobserved states (PICRUSt) software. The results showed that the community and functional structure of healthy and diseased trees differed only in the roots and needles, respectively (p < 0.05). The needles, roots, and soil formed unique bacterial community and functional structures. The abundant phyla across all samples were Proteobacteria (41.9% of total sequence), Actinobacteria (29.0%), Acidobacteria (12.2%), Bacteroidetes (4.8%), and Planctomycetes (2.1%). The bacterial community in the healthy roots was dominated by Acidobacteria, Planctomycetes, and Rhizobiales, whereas in the diseased roots, Proteobacteria, Firmicutes, and Burkholderiales were dominant. Functionally, groups involved in the cell process and genetic information processing had a higher abundance in the diseased needles, which contributed to the difference in functional structure. The results indicate that PWD can only affect the host bacteria community structure and function in certain anatomical regions of the host tree.

Comparative Transcriptome Analysis of the Heat Stress Response in Monochamus alternatus Hope (Coleoptera: Cerambycidae).

Temperature is a critical factor of insect population abundance and distribution. Monochamus alternatus Hope (Coleoptera: Cerambycidae) is a significant concern since it is transmitted vector of the pinewood nematode posing enormous economic and environmental losses. This pest shows tolerance to heat stress, especially extremely high temperatures. Exposing for 6, 12, 24, 48, or 96 h, the 50% median lethal temperatures (Ltem50) for fourth-instar larvae were 47.5, 45.5, 43.9, 43.4, and 42.3°C, respectively. A total of 63,360 unigenes were obtained from complementary DNA libraries of M. alternatus fourth-instar larvae (kept at 25°C and exposed to 40°C for 3 h) and annotated with six databases. Five hundred sixty-one genes were significantly upregulated, and 245 genes were downregulated after heat stress. The Gene Ontology enrichment analysis showed that most different expression genes are categorized into "protein folding" and "unfold protein binding" terms. In addition, "Longevity regulating pathway-multiple species," "Antigen processing and presentation" as well as "MAPK signaling pathway" were significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways. Further analysis of different expression genes showed that metabolism processes were suppressed, while ubiquitin proteolytic system, heat shock proteins, immune response, superoxide dismutase, cytochrome P450s, and aldehyde dehydrogenase were induced after heat shock. The stress signaling transduction pathways such as MAPK, Hippo, and JAK-STAT might be central convergence points in M. alternatus heat tolerance mechanism. The expression levels from quantitative real-time PCR of 13 randomly selected genes were consistent with the transcriptome results. These results showed that M. alternatus possessed strong heat tolerance and genes related to protein activity, immune response, and signal transduction composed of a complicated heat tolerance mechanism of M. alternatus. This research provided new insights into the mechanisms of thermal tolerance in other insects and aided in exploring the function of heat resistance-related genes.