Pine wilt disease (PWD) is a global quarantine disease of forests that mainly affects Pinaceae species. The disease spreads rapidly. Once infected, pine trees have an extremely high mortality rate. This paper provides a summary of the common techniques used to detect PWD, including morphological-, molecular-, chemical- and physical-based methods. By comprehending the complex relationship among pinewood nematodes, vectors and host pine trees and employing the available approaches for nematode detection, we can improve the implementation of intervention and control measures to effectively reduce the damage caused by PWD. Although conventional techniques allow a reliable diagnosis of the symptomatic phase, the volatile compound detection and remote sensing technology facilitate a rapid diagnosis during asymptomatic stages. Moreover, the remote sensing technology is capable of monitoring PWD over large areas. Therefore, multiple perspective evaluations based on these technologies are crucial for the rapid and effective detection of PWD.

Pine wilt disease was first discovered in Dongtang town, Liaoning Province, China, in 2017. However, no record of Monochamus alteratus existed in Fengcheng, where M. saltuarius is an indigenous insect, and no experimental evidence has thus far indicated that M. saltuarius can transport the Bursaphelenchus xylophilus in China. In this study, we investigated whether M. saltuarius is a vector of B. xylophilus in China. On the sixth day after eclosion, beetles began to transmit nematodes into the twigs. The transmission period of nematodes is known to be able to last for 48 days after beetle emergence. In laboratory experiments, M. saltuarius fed and transmitted B. xylophilus not only on pines but also on other non-Pinus conifers. The non-Pinus conifers preferred by M. saltuarius for feeding are Picea pungens, Picea asperata, and Abies fabri. The experimental results show that M. saltuarius functions as a vector of B. xylophilus in northeast China.

Pinus bungeana, an endangered and native coniferous tree species in China, has considerable timber and horticulture value. However, little is known about needle diseases in P. bungeana. A needle blight of P. bungeana has been observed in Hebei Province, China. P. bungeana inoculated with mycelial plugs of fungal isolates presented symptoms similar to those observed under field conditions. Ten virulent fungal isolates were identified as a small-spored Alternaria species based on morphological observations. Maximum likelihood and Bayesian phylogenetic analyses carried out with multilocus sequence typing of eight regions (SSU, LSU, ITS, gapdh, tef1, Alt a 1, endoPG, OPA10-2) assigned the pathogen to Alternaria alternata. This is the first report of A. alternata causing needle blight on P. bungeana in China.

Boxelder maple (Acer negundo L.) is widely grown as ornamental trees in China. In 2017 in Pizhou, Jiangsu Province, China, the boxelder maple trees suffered from a disease which caused wilting and yellowing leaves, rotted roots, and discolored xylem. The disease broke out from July to August. Seven fungal isolates were obtained from symptomatic tissue and tested for pathogenicity. Isolate An7 was isolated at high frequency from the discolored vascular tissues, which caused the wilt disease on healthy 2-year-old seedlings after inoculation and eventually killed all the seedlings. We conducted molecular analysis for An7, including the partial sequences of the rDNA internal transcribed spacer, calmodulin, RNA polymerase II second largest subunit and the translation elongation factor 1-α and evaluation of the variously sized conidia, chlamydospores and conidiophores and cultural traits. The pathogen was identified as Fusarium nirenbergiae, a member of the Fusarium oxysporum species complex. To our knowledge, this is the first report of F. nirenbergiae causing wilt on A. negundo in China.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

Background: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during “overwintering.” Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. Results: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. Conclusions: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.