Unraveling the Role of Cuticular Protein 3-like (HvCP3L) in the Chitin Pathway through RNAi and Methoxyfenozide Stress Response in Heortia vitessoides Moore.

Cuticle proteins (CPs) constitute a multifunctional family; however, the physiological role of Cuticle Protein 3-like (CP3L) in Heortia vitessoides Moore remains largely unclear. In this study, we cloned the HvCP3L gene from the transcriptional library of Heortia vitessoides Moore. RT-qPCR results revealed that HvCP3L exhibited high expression levels during the larval stage of Heortia vitessoides Moore, particularly at the L5D1 stage, observed in both larval and adult heads. Through RNA interference, we successfully silenced the HvCP3L gene, resulting in a significant reduction in the survival rate of Heortia vitessoides Moore, with the survival rate from larvae to adults plummeting to a mere 17.7%, accompanied by phenotypic abnormalities. Additionally, we observed that the knockdown of HvCP3L led to the inhibition of genes in the chitin pathway. Following exposure to methoxyfenozide stress, the HvCP3L gene exhibited significant overexpression, coinciding with phenotypic abnormalities. These findings underscore the pivotal role of HvCP3L in the growth and development of Heortia vitessoides Moore.

Characterization of Chitin Synthase B Gene (HvChsb) and the Effects on Feeding Behavior in Heortia vitessoides Moore.

The chitin synthase B gene is a key enzyme in the chitin synthesis of insect peritrophic matrix (PM), which affects insects' feeding behavior. The chitin synthase B gene was cloned from the transcription library of Heortia vitessoides Moore. RT-qPCR showed that HvChsb was highly expressed in the larval stage of H. vitessoides, especially on the first day of the pre-pupal stage, as well as in the midgut of larvae and the abdomen of adults. After starvation treatment, HvChsb was found to be significantly inhibited over time. After 48 h of starvation, the feeding experiment showed that HvChsb increased with the prolongation of the re-feeding time. The experimental data showed that feeding affected the expression of HvChsb. HvChsb was effectively silenced via RNA interference; thus, its function was lost, significantly decreasing the survival rate of H. vitessoides. The survival rate from larval-to-pupal stages was only 43.33%, and this rate was accompanied by abnormal phenotypes. It can be seen that HvChsb plays a key role in the average growth and development of H. vitessoides.

First report of Fusarium oxysporum Causing Fusarium Dieback on Archidendron clypearia in China.

Archidendron clypearia (Jack) Benth. (common name: Monkey-pod) is a perennial arbor tree belonging to the family Fabaceae. It is used to produce a traditional medicine to treat a variety of heat toxicity symptoms in China. The distribution of A. clypearia mainly include Guangdong, Fujian, and Zhejiang provinces in China as well as other countries in Southeast Asia. In 2018, about 30 Monkey-pod trees were observed showing typical dieback symptoms in a commercial plantation (114°36'09.401″E, 22°58'38.553″N) of Huizhou, Guangdong Province, China. In addition, white cylindrical sawdust exudates from exit holes of ambrosia beetles were observed on the trunks and main branches of the diseased trees. Discoloration was seen around the exit holes of the beetles by splitting the wood. Samples of symptomatic and asymptomatic wood tissues of branches, trunks, and roots were collected from 11 trees. To identify the pathogen, the samples were disinfected with a sodium hypochlorite solution containing 50 g/l chlorine for 60 s, rinsed three times with sterile distilled water, and placed on potato dextrose agar (PDA) for culture at 25℃ for 14 days. Beetles from exit holes of the pathogen-infected wood tissues were also collected and identified as Xyleborus affinis by their cytochrome c oxidase I (COI) sequences (GenBank accession numbers: MT921005 and MT921006) as well as morphological features. After two weeks incubation, 37 single-spore isolates were obtained (31 from galleries of branches and trunks, and 6 from mycangia and the body surface of beetles). Colonies on PDA were fusarium-like, pale orange, floccose with abundant aerial mycelia, and growing at the rate of 3.0 to 4.5 mm/d at 25℃. Conidiophores from the aerial mycelia often had single phialides. Microconidia were hyaline, 0-1 septate, (4-)6-12(-16) × 2-4 µm (n=100), Macroconidia were hyaline, ellipsoidal to falcate, 2-4 septate, (10-)25-45(-50) × 3-6 µm (n=100), with basal foot cells shaped to pointed and apical cells tapered and curved. Two representative isolates (CYML 367, 368) were used for molecular identification and pathogenicity tests. The internal transcriptional spacer region (ITS), ß-tubulin (TUB), and translation elongation factor-1α (TEF 1-α) regions were amplified with primers ITS1F/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F (Carbone and Kohn 1999) /EF-2 (O'Donnell et al.1998), respectively. DNA extraction and PCR conditions were followed the methods described by Yin et al. (2019). The amplified fragments were sequenced. The results of BLAST demonstrated that the fragment sequences of ITS (468 bp), TEF 1-α (616 bp), and TUB (295 bp) of isolates CYML367 and CYML368 had 100% similarity to correspondence sequences from the Fusarium oxysporum Schlecht. emend. Snyder & Hansen species complex (GenBank accession numbers MT032694, JF740777, and MN451171, respectively). Sequences generated from this study were deposited into GenBank under accession numbers MN826824 and MN826825 (ITS), MN839680 and MN839681 (TUB), and MN839682 and MN839683 (TEF 1-α). Pathogenicity tests were conducted on 2-year-old seedlings (60-70 cm height, 1.0-1.2 cm in diameter) of A. clypearia with the representative isolates. Ten seedlings were inoculated with each isolate. The stem surface was disinfected with 75% ethanol for 30 s, and rinsed with sterilized water, wounded by removing part of the phloem and xylem, and placed with a mycelial plug (5 mm diameter) from the margin of a 7-day-old PDA plate with mycelia facing the cambium. The inoculated wounds were wrapped with a medical sterile gauze to prevent desiccation and contamination. Control plants were inoculated with non-colonized PDA plugs. These inoculated and control plants were incubated in a chamber at 25℃, 50% humidity, 12 h light/12 h dark cycle. After 3 weeks, all inoculated plants displayed Fusarium dieback symptoms similar to those observed on the original diseased trees in the plantation. The average lesion lengths (1.2/1.7 cm) caused by two isolates were all significantly longer than the wounds in the negative controls (P<0.05). The same fungus was re-isolated from the symptomatic plants, thus fulfilling the Koch's postulates. Further studies should be conducted on the potential vector association, transmission route, and management strategies of this pathogen on A. clypearia in growing regions of China. To our best knowledge, this is the first report of F. oxysporum causing dieback on A. clypearia in China.

Phylogenetic re-evaluation of the Grosmannia penicillata complex (Ascomycota, Ophiostomatales), with the description of five new species from China and USA.

The Grosmannia penicillata complex (Ophiostomatales, Ascomycota) is one of the major species complexes in Leptographium sensu lato. Most of these are wood staining fungi associated with conifer-infesting bark beetles, and the complex encompasses the type species of the genus Grosmannia. Yet the phylogenetic relationships of species within the complex is unresolved. The aim of this study was to re-evaluate the circumscriptions of all known species in the G. penicillata complex, as well as isolates resembling G. penicillata obtained from a recent survey in China. Phylogenetic analyses of four gene regions: Internal transcribed spacer 2 and large subunit (ITS2-LSU), beta-tubulin (TUB), calmodulin (CAL), and translation elongation factor 1 alpha (TEF-1α) resolved the relationships of 15 species, including four new species (Grosmannia xianmiense sp nov., Grosmannia purpurea sp. nov., Grosmannia crassifolia sp. nov. and Grosmannia maixiuense sp. nov.), from China. Some isolates from pine in the USA that had previously been identified as Grosmannia abietina, represented a distinct taxon that is described here as Grosmannia xeno-abietina sp. nov.

Taxonomy and phylogeny of the Leptographium olivaceum complex (Ophiostomatales, Ascomycota), including descriptions of six new species from China and Europe.

The Leptographium olivacea complex encompasses species in the broadly defined genus Leptographium (Ophiostomatales, Ascomycota) that are generally characterized by synnematous conidiophores. Most species of the complex are associates of conifer-infesting bark beetles in Europe and North America. The aims of this study were to reconsider the delineation of known species, and to confirm the identity of several additional isolates resembling L. olivacea that have emerged from recent surveys in China, Finland, Poland, Russia, and Spain. Phylogenetic analyses of sequence data for five loci (ACT, TUB, CAL, ITS2-LSU, and TEF-1α) distinguished 14 species within the complex. These included eight known species (L. cucullatum, L. davidsonii, L. erubescens, L. francke-grosmanniae, L. olivaceum, L. olivaceapini, L. sagmatosporum, and L. vescum) and six new species (herein described as L. breviuscapum, L. conplurium, L. pseudoalbum, L. rhizoidum, L. sylvestris, and L. xiningense). New combinations are provided for L. cucullatum, L. davidsonii, L. erubescens, L. olivaceum, L. olivaceapini, L. sagmatosporum and L. vescum. New Typifications: Lectotypes are designated for L. olivaceum, L. erubescens and L. sagmatosporum. Epitypes were designated for L. olivaceapini and L. sagmatosporum. In addition to phylogenetic separation, the synnematous asexual states and ascomata with almost cylindrical necks and prominent ostiolar hyphae, distinguish the L. olivaceum complex from others in Leptographium.

Multigene phylogenies and morphological characterization of five new Ophiostoma spp. associated with spruce-infesting bark beetles in China.

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark beetles (Coleoptera: Scolytinae). Some of these are serious tree pathogens, while the majority is blue-stain agents of timber. In recent years, various bark beetle species have been attacking spruce forests in Qinghai province, China, causing significant damage. A preliminary survey was done to explore the diversity of the ophiostomatoid fungal associates of these beetles. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with spruce-infesting bark beetles in Qinghai Province, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Chinese isolates, using multigene phylogenetic analyses. Results obtained from four gene regions (ribosomal internal transcribed spacer regions, ß-tubulin, calmodulin, translation elongation factor-1α) revealed five new Ophiostoma spp. from Qinghai. These included O. nitidus sp. nov., O. micans sp. nov., and O. qinghaiense sp. nov. in a newly defined O. piceae complex. The other two new species, O. poligraphi sp. nov. and O. shangrilae sp. nov., grouped in the O. brunneo-ciliatum complex. Based on DNA sequence and morphological comparisons, we also show that O. arduennense and O. torulosum are synonyms of O. distortum, while O. setosum is a synonym of O. cupulatum.

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, ß-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.