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European hornbeam (Carpinus betulus L.) is widely planted in landscaping. In October 2021 and August 2022, leaf spot was observed on C. betulus in Xuzhou, Jiangsu Province, China. To identify the causal agent of anthracnose disease on C. betulus, 23 isolates were obtained from the symptomatic leaves. Based on ITS sequences and colony morphology, these isolates were divided into four Colletotrichum groups. Koch’s postulates of four Colletotrichum species showed similar symptoms observed in the field. Combining the morphological characteristics and multi-gene phylogenetic analysis of the concatenated sequences of the internal transcribed spacer (ITS) gene, Apn2-Mat1-2 intergenic spacer (ApMat) gene, the calmodulin (CAL) gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, Glutamine synthetase (GS) gene, and beta-tubulin 2 (TUB2) genes, the four Colletotrichum groups were identified as C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense. This study is the first report of four Colletotrichum species causing leaf spot on European hornbeam in China, and it provides clear pathogen information for the further evaluation of the disease control strategies.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a notorious parasitic nematode of pine trees that causes pine wilt disease (PWD), leading to extensive mortality of different pine species around the world and considerable economic losses, thus posing a threat to healthy pines worldwide. Fast and accurate detection technology is necessary for the management of PWD spread. This study describes the development of a new DNA extraction method and detection technology, enzyme-mediated amplification (EMA), using primers and a newly designed probe according to the rRNA internal transcribed spacer gene ITS2. The detection process can be completed within 40 min, including DNA extraction for 10 min and detection for 30 min, by exploiting the synergistic action of multiple enzymes. This method can detect PWNs from different geographic areas quickly and accurately at all life stages, singly or in a mixture, and can distinguish PWNs from other species of the Bursaphelenchus group, showing that it is not only reliable but also rapid, greatly improving the efficiency and speed of PWN detection. Therefore, the technology is expected to be highly beneficial in PWN quarantine testing.

Cyclocarya paliurus is an endemic Chinese tree species with considerable medicinal, timber, and horticultural value. The anthracnose disease of C. paliurus is caused by the fungal pathogen Colletotrichum fructicola, which results in great losses in yield and quality. Here, resistance evaluation of six cultivars of C. paliurus exhibited varying degrees of resistance to C. fructicola infection, where Wufeng was the most resistant and Jinggangshan was the most susceptive. Physiological measurements and histochemical staining assays showed that the Wufeng cultivar exhibits intense reactive oxygen species accumulation and defense capabilities. A multiomics approach using RNA sequencing and metabolome analyses showed that resistance in C. paliurus (Wufeng) is related to early induction of reprogramming of the flavonoid biosynthesis pathway. In vitro antifungal assays revealed that the flavonoid extracts from resistant cultivars strongly inhibited C. fructicola hyphal growth than susceptible cultivars. Relative gene expression analysis further demonstrated the pivotal antifungal role of C. paliurus flavonoids in targeting Colletotrichum appressorium formation. Together, these results represent a novel resistance mechanism of C. paliurus against anthracnose through the reprogramming of flavonoids, which will lay a foundation for breeding anthracnose-resistant varieties and the application of flavonoid extraction of C. paliurus as a natural antifungal treatment.

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.

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.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.