Chitin Synthase Research Articles

Sequential host-bacteria and bacteria-bacteria interactions determine the microbiome establishment of Nematostella vectensis

BackgroundThe microbiota of multicellular organisms undergoes considerable changes during host ontogeny but the general mechanisms that control community assembly and succession are poorly understood. Here, we use bacterial recolonization experiments in Nematostella vectensis as a model to understand general mechanisms determining bacterial establishment and succession. We compared the dynamic establishment of the microbiome on the germfree host and on inert silicone tubes.ResultsFollowing the dynamic reconstruction of microbial communities on both substrates, we show that the initial colonization events are strongly influenced by the host but not by the silicone tube, while the subsequent bacteria-bacteria interactions are the main driver of bacterial succession. Interestingly, the recolonization pattern on adult hosts resembles the ontogenetic colonization succession. This process occurs independently of the bacterial composition of the inoculum and can be followed at the level of individual bacteria. To identify potential metabolic traits associated with initial colonization success and potential metabolic interactions among bacteria associated with bacterial succession, we reconstructed the metabolic networks of bacterial colonizers based on their genomes. These analyses revealed that bacterial metabolic capabilities reflect the recolonization pattern, and the degradation of chitin might be a selection factor during early recolonization of the animal. Concurrently, transcriptomic analyses revealed that Nematostella possesses two chitin synthase genes, one of which is upregulated during early recolonization.ConclusionsOur results show that early recolonization events are strongly controlled by the host while subsequent colonization depends on metabolic bacteria-bacteria interactions largely independent of host ontogeny.-JTs9knfCQzALX-coLcUGoVideo

Transient septin sumoylation steers a Fir1-Skt5 protein complex between the split septin ring.

Ubiquitylation and phosphorylation control composition and architecture of the cell separation machinery in yeast and other eukaryotes. The significance of septin sumoylation on cell separation remained an enigma. Septins form an hourglass structure at the bud neck of yeast cells that transforms into a split septin double ring during mitosis. We discovered that sumoylated septins recruit the cytokinesis checkpoint protein Fir1 to the peripheral side of the septin hourglass just before its transformation into the double-ring configuration. As this transition occurs, Fir1 is released from the septins and seamlessly relocates between the split septin rings through synchronized binding to the scaffold Spa2. Fir1 binds and carries the membrane-bound Skt5 on its route to the division plane where the Fir1-Skt5 complex serves as receptor for chitin synthase III.

Systematic assessment of the antifungal mechanism of soil fumigant methyl isothiocyanate against Fusarium oxysporum

Fusarium oxysporum is an important phytopathogenic fungus, it can be controlled by the soil fumigant methyl isothiocyanate (MITC). However, the antimicrobial mechanism of MITC against F. oxysporum, especially at the transcriptional level, is still unclear. In this experiment, the antimicrobial mechanism of MITC against F. oxysporum was investigated. Our results indicated that when F. oxysporum was exposed to 6 mg/L MITC for 12 h, the inhibitory rate of MITC on F. oxysporum was 80%. Transmission electron microscopes showed that the cell wall and membrane of F. oxysporum had shrunk and folded, vacuoles increased, and mitochondria swelled and deformed. In addition, the enzyme activity of F. oxysporum treated with MITC showed a decrease of 32.50%, 8.28% and 74.04% in catalase, peroxidase and superoxide dismutase, respectively. Transcriptome sequencing of F. oxysporum was performed and the results showed that 1478 differentially expressed genes (DEGs) were produced in response to MITC exposure. GO and KEGG analysis showed that the DEGs identified were involved in substance and energy metabolism, signal transduction, transport and catalysis. MITC disrupted cell homeostasis by influencing the expression of some key genes involved in chitin synthase and detoxification enzymes production, but F. oxysporum also protected itself by up-regulating genes involved in energy synthesis (such as upregulating acnA, CS and LSC2 in TCA). qRT-PCR data validated the reliability of transcriptome data. Our research used biochemical and genetic techniques to identify molecular lesions in the mycelia of F. oxysporum exposed to MITC, and provide valuable insights into the toxic mechanism of pathogenic fungi mediated by MITC. These techniques are also likely to be useful for rapidly screening and identifying new, environmentally-friendly soil fumigants that are efficacious against fungal pathogens.

MicroRNA-989 controls Aedes albopictus pupal-adult transition process by influencing cuticle chitin metabolism in pupae

BackgroundAedes albopictus is a vector of numerous devastating arboviruses and places heavy burdens on global public health. Chitin is one of the important components of cuticles and targeting chitin metabolism is a promising strategy for preventing mosquito dispersal and mosquito-borne diseases. Increasing evidence suggests that microRNAs (miRNAs) play crucial roles in various physiological processes of insects.MethodsA previous analysis suggested that the microRNA miR-989 is potentially involved in chitin metabolism in Ae. albopictus pupae. In the present study, we found that the expression level of miR-989 was significantly overexpressed after injection of agomir. A dual-luciferase assay was used to determine the direct target of miR-989. Survival rate, eclosion rate and malformation rate were statistically analyzed to evaluate the potential effect of miR-989. Hematoxylin–eosin staining and chitin staining were used to evaluate the microstructural changes in the cuticles of Ae. albopictus pupae.ResultsOverexpression of miR-989 resulted in a significantly reduced survival rate and eclosion rate of pupae and an elevated malformation rate of adults. The results suggested that miR-989 acted as a regulator of chitin metabolism in Ae. albopictus pupae by affecting the transcript levels of the Ae. albopictus genes encoding chitin synthase 1 (AaCHS1) and chitinase 10 (AaCht10). The altered expression levels of the two chitin metabolism-related enzymes (CHS1 and Cht10, respectively) caused the structural changes in cuticles and further affected the pupal-adult transition process of Ae. albopictus. XM_029863591.1 was proven to be the target gene of miR-989 and displayed similar effects on pupae as miR-989.ConclusionsThe microRNA miR-989 was found to be essential for chitin metabolism in old and new cuticles of Ae. albopictus pupae. The results of the current study suggested that miR-989 could be used as a potential target to control Ae. albopictus.Graphical

Occurrence of leaf spot caused by Colletotrichum plurivorum on cowpea in Fujian Province, China

Cowpea (Vigna unguiculata (L.) Walp.) is cultivated worldwide for vital food source and vegetable. During May to October 2021, a serious leaf spot disease occurred on cowpea in Pingnan prefecture, Fujian Province, China. The disease infected more than 60% of the cowpea leaves under natural conditions. The causal organism was recovered from diseased leaves exhibiting characteristic symptoms of brown spots, and was identified as Colletotrichum plurivorum Damm, Alizadeh & Toy. Sato according to morphological features and sequencing of the ribosomal DNA internal transcribed spacer (ITS), partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and beta-tubulin (TUB2) gene sequences. Pathogenic assays were carried out using cowpea ‘Yaoqianshu’ at the five-to eight-leaf stage. The results indicated that all tested C. plurivorum isolates could infect healthy cowpea leaves, even in the lack of wounds, and cause leaf spot symptoms similar to originally observed on cowpea leaves in the fields. Finally, the identical isolates were successfully re-isolated from those of inoculated leaves. To our knowledge, this is the first report of C. plurivorum causing anthracnose on cowpea in Fujian Province, China.

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate)

We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches.

First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 μm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Iris lactea in Daqing, China.

Iris lactea Pall. has high ornamental value and strong drought resistance, which can be used as a useful sand-stabilization and ornamental plant. In September 2021, typical anthracnose symptoms on I. lactea were found in the campus of Heilongjiang Bayi Agricultural University (125°10'45.31"E, 46°35'36.88"N), Daqing, China. Disease incidence varied from 37 to 51% at four survey sites (~20 m2 per site) of approximately 1,000 plants. Initially brown lesions with a yellow halo, and gray in the center were observed on the leaves. As disease progressed, the lesions expanded rapidly, resulting in dieback. To identify the pathogen, symptomatic tissues were excised from four infected leaves of four individual plants, surface sterilized for 1 min in 75% ethanol, washed twice with sterile water, plated on potato dextrose agar (PDA) contain 0.5 g/L streptomycin sulfate, and incubated at 25℃ for 3 to 4 days under dark conditions. Four morphologically similar fungal isolates (Irs-1 to -4) were obtained by using a single-spore isolation. After growth on PDA for 17 days at 25℃, a circular like zonation colonies with a color of greyish to gray green were observed. On the reverse side, circular like with zig-zag zonation colonies light brown to light black from the margin to the center. Conidiophores were pale brown, septate. Conidia were hyaline, aseptate, curved or slightly curved, round, or somewhat acute apex, base truncate, unicellular, whose sizes ranged at 18.5 ± 2.3 × 4.6 ± 0.7 μm (n = 170), with length/width ratio 4.04. Appressoria were in oval, star, or irregular shape, with brown color with sizes approximately at 13.3 ± 2.1 × 10.4 ± 2.1 μm (n = 30). These morphological characteristics are consistent with Colletotrichum spaethianum with curved conidia (Damm et al. 2009). For molecular identification, primers ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/ACT-783R and CHS-354R/CHS-79F (Carbone and Kohn 1999) were used to amplify the partial region of rDNA-ITS, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), and chitin synthase 1 (CHS-1) from fresh mycelia of the four isolates (Irs-1 to -4), respectively. The sequences were submitted to GenBank (ITS: OM004553, OP804624, OR029392, and OR029391; GAPDH: OP883938, OP883939, OR060963, and OR060962; ACT: OP883940, OP883941, OR060965, and OR060964; CHS: OP868844, OP868845, OR046504, and OR046503). Phylogenetic analysis based on the ITS, GAPDH, ACT and CHS gene sequences indicated that isolates obtained in this study were all clustered with C. spaethianum. Pathogenicity test was conducted by inoculating mycelial plugs on the I. lactea seedlings (2 seedlings with 4 to 5 leaves per isolate). Two seedlings inoculated with sterilized PDA plugs were used as control. All inoculated plants were maintained in humid chamber at 25℃ under dark, and typical anthracnose symptoms were observed up to 6 days after inoculation, while the control leaves were asymptomatic. The same pathogen was successfully re-isolated and phenotypically identical to the original isolates to fulfill Koch's postulates. C. spaethianum has been described on Hemerocalis flava (Vieira and Michereff 2014) and Allium fistulosum (Santana et al. 2016) in Brazil, Polygonatum cyrtonema in China (Ma et al. 2020), Iris germanica in Japan (Sato et al. 2012). To our knowledge, this is the first report of C. spaethianum causing anthracnose disease on I. lactea in Daqing, China.

First Report of Anthracnose Caused by Collectotrichum higginsianum on Rumex crispus in China.

Rumex crispus L. is a perennial herb with medicinal properties derived from its roots and whole plant (Bhandari et al. 2022). In December 2022, symptoms of anthracnose were observed in approximately 40% of naturally occurring R. crispus plants in Longquan Reservior, Nanchang city (115°53' N, 28°43' E), Jiangxi Province, China. Initially, red lesions appeared randomly on various parts of the leaf blade, which gradually became dry and brown at the center, eventually leading to leaf death. To isolate the fungal pathogen responsible, ten symptomatic leaves were randomly collected and their lesions were cut into small pieces (4 × 4 mm). The leaf fragments were surface-sterilized in 70% ethanol for 45 s and then in 1% NaClO for 45 s. The leaf pieces were rinsed three times with sterile distilled water. The surface-sterilized leaf pieces were then placed onto potato dextrose agar (PDA) and incubated at 28 ℃, dark condition for 3 days. Twelve isolates were obtained, characterized by a milky white and uneven growth pattern with a white root-like structure branching out at the edge, along with scattered black deposits on the bottom of the plate. Conidiogenous cells cylindrical, smooth-walled, hyaline, 9.3-23.2 × 3.6-4.2 μm. Conidia elliptical, aseptate, smooth-walled, with one end blunt and the other truncate, ranging in size from 10.4 to 22.3 (mean 16.7) μm in length and 3.2 to 5.0 (mean 4.1) μm in width (n = 50), which are consistent with the characteristics of the members of Colletotrichum destructivum species complex (Damm et al. 2014). To accurately identify the strain, three representative isolates, namely JFRL 03-930, JFRL 03-931, and JFRL 03-935, were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GADPH), histone3 (HIS3), and beta-tubulin (TUB2) genes were amplified and sequenced using specific primer pairs, including ITS5/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, GYLH3F/CYLH3R, and T1/Bt2b (Damm et al. 2014). All sequences were deposited in GenBank with accession numbers OQ560476-OQ560478 (ITS), OQ576154-OQ576156 (ACT), OQ576157-OQ576159 (CHS), OQ576160-OQ576162 (HIS3), OQ576163-OQ576165 (GADPH), and OQ576166-OQ576168 (TUB2). A maximum likelihood phylogenetic tree was constructed using IQtree v1.5.6 based on the combined ITS, ACT, CHS, GAPDH, HIS3 and TUB2 data set (Nguyen et al. 2015), The phylogenetic tree showed that the three isolates clustered with C. higginsianum in a clade with 91% bootstrap support. Based on morphology and molecular characters, the isolates were identified as C. higginsianum of the C. destructivum species complex. To confirm the pathogenicity, One-year-old R. crispus were collected from the wild and potted in an climate chamber. Six healthy leaves of R. crispus were surface sterilized with 70% ethanol and wounded by sterile needle, and a 20-μl conidial suspension (3×105 conidia/ml) of the isolate JFRL 03-931 was inoculated on the wound. Another set of six leaves of R. crispus was inoculated with distilled water as controls. The potted plants were incubated under conditions of 25 ℃ and 80% humidity. After 10 days, reddish brown spots were observed on all inoculated leaves, while the control leaves remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated leaves and confirmed as C. higginsianum by morphological and molecular analysis. It has been reported that C. higginsianum caused anthracnose disease on several cruciferous vegetables, Boehmeria nivea and Rumex acetosa in China (Damm et al. 2014; Wang et al. 2011; Patel et al. 2014; Zhang et al. 2018). But to our knowledge, this is the first report of C. higginsianum casued anthracnose on Rumex crispus in China. Therefore, we should pay more attention to this pathogen and develop appropriate control strategies.

Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.

Histochemistry and transcriptomics of mucins and peritrophic membrane (PM) proteins along the midgut of a beetle with incomplete PM and their complementary function

The midgut of Zabrotes subfasciatus (Coleoptera) and other insects may have regions lacking a peritrophic membrane (matrix, PM) and covered with a jelly-like material known as peritrophic gel. This work was undertaken to test the hypothesis that the peritrophic gel is a vertebrate-like mucus. By histochemistry we identified mucins along the whole midgut, which contrasts with the known occurrence of PM only at the posterior midgut. We also analyzed the expression of the genes coding for mucus-forming mucins (Mf-mucins), peritrophins, chitin synthases and chitin deacetylases along the midgut and carcass (insect without midgut) by RNA-seq. Mf-mucins were identified as proteins with high O-glycosylation and multiple tandem repeats of Pro/Thr/Ser residues. Peritrophins were separated into PM proteins, cuticular proteins analogous to peritrophins (CPAPs) and ubiquitous-chitin-binding domain-(CBD)-containing proteins (UCBPs). PM proteins have at least 3, CPAP one or 3, and UCBPs have a varied number of CBDs. PM proteins are more expressed at midgut, CPAP at the carcass, and UCBP at both. The results showed that most PM proteins are mainly expressed at the posterior midgut, together with midgut chitin synthase and chitin deacetylase, and in agreement with the presence of PM only at the posterior midgut by visual inspection. The excretion of most midgut chitinase is avoided, suggesting that the shortened PM is functional. Mf-mucins are expressed along the whole midgut, probably forming the extracellular mucus layer observed by histochemistry. Thus, the lack of PM at anterior and middle midgut causes the exposure of a mucus, which may correspond to the previously described peritrophic gel. The putative functional interplay of mucus and PM is discussed. The major role of mucus is proposed to be tissue protection and of PM to enhancing digestive efficiency by allowing enzyme recycling.

Effects of β-1,3-glucan on growth, immune responses, and intestinal microflora of the river prawn (Macrobrachium nipponense) and its resistance against Vibrio parahaemolyticus

In this study, we investigated the impact of β-1,3-glucan on the immune responses and gut microbiota of the river prawn (Macrobrachium nipponense) in the presence of Vibrio parahaemolyticus stress. Shrimps were fed one of the following diets: control (G1), 0.2% curdlan (G2), 0.1% β-1,3-glucan (G3), 0.2% β-1,3-glucan (G4), or 1.0% β-1,3-glucan (G5) for 6 weeks and then challenged with V. parahaemolyticus for 96 h. Under Vibrio stress, shrimps in G4 exhibited the highest length gain rate, weight gain rate, and survival rate. They also showed increased intestinal muscle thickness and villus thickness compared to the control and 0.2% curdlan groups. The apoptosis rate was lower in G4 than in the control group, and the digestive enzyme activities (pepsin, trypsin, amylase, and lipase), immune enzyme activities (acid phosphatase, alkaline phosphatase, lysozyme, and phenoxidase), and energy metabolism (triglyceride, cholesterol, glycogen, and lactate dehydrogenase) were enhanced. Expression levels of growth-related genes (ecdysone receptor, calmodulin-dependent protein kinase I, chitin synthase, and retinoid X receptor) and immune-related genes (toll-like receptor 3, myeloid differentiation primary response 88, mitogen-activated protein kinase 7, and mitogen-activated protein kinase 14) were higher in G4 than in the control. Microbiota analysis indicated higher bacterial abundance in shrimps fed β-1,3-glucan, as evidenced by Sob, Chao1, and ACE indices. Moreover, 0.2% β-1,3-glucan increased the relative abundances of Bacteroidota and Firmicutes while reducing those of Corynebacteriales and Lactobacillales. In summary, β-1,3-glucan enhances immune enzyme activities, alters immune-related gene expression, and impacts gut microbial diversity in shrimp. These findings provide valuable insights into the mechanisms underlying β-1,3 glucan's immune-enhancing effects.

First Report of Anthracnose Caused by Colletotrichum ciggaro on Lonicera macranthoides Hand.-Mazz. in China.

Lonicera macranthoides Hand.-Mazz. is a traditional medicinal plant that is cultivated in Hunan, Yunnan, and Guizhou Provinces in China. In June 2020, a new leaf spot disease was observed on this plant in Longhui County, Shaoyang City, Hunan Province, China, where 14,000 hm2 of L. macranthoides had been planted. About 20% of the total cultivated area exhibited symptoms. Brown spots appeared on the leaves during the early stage and gradually expanded into irregular lesions, which became necrotic and dry. The whole plant withered and died in severe cases. To isolate the pathogen, the infected leaves were collected from different fields and washed with flowing sterile water. The small lesions were then cut and surface sterilized with 75% alcohol for 45 s followed by a 3 min treatment in 3% sodium hypochlorite. The lesions were rinsed five times in sterile water, incubated on potato dextrose agar (PDA) plates and cultured for 3-5 d at 28℃. In total, eleven isolates were obtained, and eight of them were Colletotrichum (isolation frequency 73%). Three representative isolates (JYH1, JYH2, and JYH5) were selected for further study. The fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were aseptate, transparent, cylindric, and thin walled, which measured 11.54 to 22.64 × 3.55 to 4.75 μm (n=100). Six genetic regions were amplified and sequenced to further confirm the identity of fungus. They included β-tubulin (TUB2), the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences were submitted to GenBank (ITS: OQ746331; ON954583; OQ746334; TUB2: OQ772278; ON960155; OQ772279; CHS: OQ772280; ON960156; OQ772281; ACT: OQ772282; ON960157; OQ772283; CAL: OQ772284; ON960158; OQ772285; GAPDH: OQ772286; ON960159; OQ772287). The construction of a 6-gene joint phylogenetic tree analysis showed that the three isolates unambiguously clustered with Colletotrichum kahawae subsp. ciggaro strains C022-1 (GenBank: KJ001120.1, KJ001124.1, KJ001109.1, KJ001102.1, KJ001106.1, KJ001113.1) and R019 (GenBank: JN715847.1, KC860023.1, KC859980.1, KC859954.1, KC859972.1, KC859997.1), which was recently reclassified as C. cigarro (Cabral et al. 2020). Three representative isolates were used for the pathogenicity test on the young leaves of the whole plant. A sterile pin was used to prick the leaf epidermis, and 6 × 6 mm mycelial blocks that had been cultured on PDA for 7 d were placed on the leaf wounds. The controls were treated in the same manner except that sterile blocks of PDA were used. There were three replicates per treatment. All the plants used in the experiment were maintained at 28°C in a climate chamber. There was a 12 h photoperiod, and the chamber was kept at 80% relative humidity. Dark brown spots appeared at the sites of inoculation on the plants after 5 days. All the strains that were re-isolated from the lesions shared the same morphological characteristics and had the same type of colonies as the pathogen Colletotrichum ciggaro. Thus, Koch's postulates were fulfilled. C. ciggaro had been shown to cause anthracnose on Olea europaea L. (Weir et al. 2012), Mangifera indica L. (Ismail et al. 2015), Citrus reticulata L. (Perrone et al. 2016) and Areca catechu L. (Zhang et al. 2020). To our knowledge, this is the first report of C. ciggaro causing anthracnose on L. macranthoides in China and worldwide. This research provides a basis for further research to control epidemics of this disease.

Dalbergia odorifera, a new host of Colletotrichum tropicale causing anthracnose in China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is an endangered, wild and second class key protected tree species in China. It has been cultivated widely in south China because of its valuable rosewood and pharmacological uses. During January and September 2022, anthracnose was observed on D. odorifera in Chikan, Zhanjiang, Guangdong (N21°15' 9.4" E110°22' 25.9"). All of the D. odorifera plants were infected and the leaf disease incidence was above 30%. The lesions were 0.35 to 0.65 cm in size, greyish-white, and gradually expanded into brown black circular spots surrounded by a yellowish halo. Later, the centers of spots were white with dark-brown borders. In severe cases, the leaves turned yellow, withered and fell off. To isolate the pathogen, about 5×5 mm tissues at the disease-health junction of spots were sterilized with 75% ethanol for 30s and 3% hydrogen peroxide for 3 min, rinsed with sterile water three times, and plated onto PDA for incubation at 25-28℃ in the dark. Isolates were obtained by picking the tip of hypha growing from the tissues, further purified by coating the diluted spore suspension to obtain single-spore isolates. Colonies with abundant aerial mycelium were white to light gray, 4.0 to 5.0 cm in diameter after 5 days at 25 to 28℃ in the dark. Conidiogenous cells were hyaline, cylindrical and monoblastic. Conidia were unicellular, subcylindrical, hyaline, guttulate, 3.5 to 5.6×11.1 to 16.8 (av. 4.6±0.48 × 14.0±1.25) μm (n>50). Appressoria were dark brown subglobose, clavate, fusiform, occasionally lobed, terminal, 7.8 to 16.3×4.6 to 8.9 μm. No swollen cells and setae were observed. To further confirm identity of the fungus, partial regions of six genetic loci of isolate ACCC 35246 (stored in Agricultural Culture Collection of China) were amplified and sequenced: actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), histone (HIS), beta-tubulin (TUB) and internal transcribed spacer (ITS) utilizing the primer pairs (Damm et al. 2012; Weir et al. 2012; White et al 1990) (OP314900-OP314904 and OP269660, respectively). Based on a BLAST analysis, the six sequences were about 99% identical to those of Colletotrichum tropicale holotype strain CBS124949 (ACT: JX009489 270/272; CHS: JX009870 295/299; GPDH: JX010007 274/279; HIS: KY856395 638/670; ITS: JX010264 546/548 and TUB: JX010407 494/497, respectively) (Weir et al. 2012). Phylogenetic analyses, using a combined dataset of ACT, CHS, GPDH, ITS and TUB were carried out in MEGA X using the maximum likelihood method, placed the fungus within the C. tropicale clade. Based on morphogical and molecular analyses, the fungus was identified as C. tropicale (Rojas et al 2010). To test the pathogenicity, five healthy leaves of 1 year-old potted plants were stab-wounded with a sterile needle and inoculated with 10 µL of spore suspension (105 conidia/mL). Another five healthy leaves were inoculated with sterile water as the control. Symptoms expressed by the inoculated leaves, 10 days after inoculation, were the same as those of the diseased plants, while the non-inoculated leaves remained asymptomatic. The same fungus based on morphogical and molecular criteria was re-isolated from the spots. As an endophyte or plant pathogen, C. tropicale inhabits a wide host range of plant species belonging to 31 genera in 25 families (Farr and Rossman 2022), D. odorifera is a new host of C. tropicale causing anthracnose in China. This work will be helpful for the diagnosis and control of this disease.

Effects of dietary melatonin on antioxidant and immune function of the Pacific white shrimp (Litopenaeus vannamei), as determined by transcriptomic analysis

Melatonin (MT) is regarded as an antioxidant and immunostimulant that can efficiently scavenge free radicals and activate antioxidant enzymes. The aim of this study was to investigate the effects of dietary MT on the growth performance and immune function of the Pacific white shrimp (Litopenaeus vannamei). Six groups of L. vannamei were supplemented with dietary MT at 0, 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg levels for 2 months. RNA-Seq analysis was performed to obtain transcriptome data of the control group and the group supplemented with dietary MT at 82.7 mg/kg BW. In total, 1220 DEGs (799 up-regulated and 421 down-regulated) were identified. Pathways and genes related to growth performance and immune function were verified by real-time quantitative polymerase chain reaction. The total hemocyte count, phagocytosis rate, and respiratory burst were significantly increased in the MT (82.7 mg/kg BW) group as compared to the control group. Analysis of antioxidant-related enzymes in the hepatopancreas showed that dietary MT (82.7 mg/kg BW) significantly increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, while dietary MT at 41.2 mg/kg BW significantly increased activities of glutathione S-transferase, lysozyme (LZM), and phenoloxidase (PO). At the transcriptional level, dietary MT up-regulated expression levels of genes associated with antioxidant immunity and growth, which included PO, SOD, LZM, GPx, chitin synthase, ecdysone receptor, calcium–calmodulin dependent protein kinase I, and retinoid X receptor. In conclusion, dietary MT may improve the growth performance and immune function of L. vannamei to some extent.

Osmotic Stress Responses, Cell Wall Integrity, and Conidiation Are Regulated by a Histidine Kinase Sensor in Trichoderma atroviride.

Trichoderma atroviride responds to various environmental stressors through the mitogen-activated protein kinase (MAPK) Tmk3 and MAPK-kinase Pbs2 signaling pathways. In fungi, orthologues to Tmk3 are regulated by a histidine kinase (HK) sensor. However, the role of T. atroviride HKs remains unknown. In this regard, the function of the T. atroviride HK Nik1 was analyzed in response to stressors regulated by Tmk3. The growth of the Δnik1 mutant strains was compromised under hyperosmotic stress; mycelia were less resistant to lysing enzymes than the WT strain, while conidia of Δnik1 were more sensitive to Congo red; however, ∆pbs2 and ∆tmk3 strains showed a more drastic defect in cell wall stability. Light-regulated blu1 and grg2 gene expression was induced upon an osmotic shock through Pbs2-Tmk3 but was independent of Nik1. The encoding chitin synthases chs1 and chs2 genes were downregulated after an osmotic shock in the WT, but chs1 and chs3 expression were enhanced in ∆nik1, ∆pbs2, and ∆tmk3. The vegetative growth and conidiation by light decreased in ∆nik1, although Nik1 was unrequired to activate the light-responsive genes by Tmk3. Altogether, Nik1 regulates responses related to the Pbs2-Tmk3 pathway and suggests the participation of additional HKs to respond to stress.

Morphological and pathological characterization ofColletotrichumspecies causing anthracnose of litchi leaves in Guangxi, China

AbstractLitchi (Litchi chinensisSonn.) is an evergreen subtropical fruit tree native to southern China. Litchi is vulnerable to a wide range of diseases affecting yield and fruit quality. Anthracnose is one of the main diseases during the period of growth and storage, which has a serious impact on the quality and production of litchi. In December 2020 to May 2021, typical anthracnose symptoms were observed on litchi leaves in different orchards in Qinzhou City, Guangxi Province, Southern China. According to colony features, conidial and appressorial morphology, and sequence analysis of several genomic regions (internal transcribed spacer (ITS) region, chitin synthase (chs‐1), actin (act), calmodulin (cal), glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), β‐tubulin (tub2) and the intergenic region of apn2 and MAT1‐2‐1 (ApMat)), 44 isolates were obtained, and 26 were identified as threeColletotrichumspecies:C. fructicola(50%),C. siamense(42.31%),C. gigasporum(7.69%). The pathogenicity tests were performed with conidial suspension and mycelia plugs to inoculate wounded litchi seedlings. The results of pathogenicity tests showed that the virulence ofC. gigasporumwas the weakest, and the virulence ofC. fructicolawas the strongest. This is the first report ofC. gigasporumcausing anthracnose of litchi worldwide.

Two new species of Colletotrichum (Glomerellaceae, Glomerellales) causing walnut anthracnose in Beijing.

Colletotrichum species are plant pathogens, saprobes and endophytes on various plant hosts. It is regarded as one of the 10 most important genera of plant pathogens in the world. Walnut anthracnose is one of the most severe diseases affecting walnut productivity and quality in China. In this study, 162 isolates were obtained from 30 fruits and 65 leaf samples of walnut collected in Beijing, China. Based on morphological characteristics and DNA sequence analyses of the concatenated loci, namely internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1) and beta-tubulin (TUB2), these isolates were identified as two novel species of Colletotrichum, i.e. C.juglandicola and C.peakense. Koch's postulates indicated that both C.juglandicola and C.peakense could cause anthracnose in walnut.

First Report of Colletotrichum siamense Causing Anthracnose on Dioscorea alata in China.

Dioscorea alata is an annual or perennial dicotyledonous plant which is a vegetatively propagated tuberous food crop (Mondo et al. 2021). In 2021, symptoms of leaf anthracnose occurred on D. alata plants at a plantation in Changsha, the Hunan Province of China (28°18' N; 113°08'E). Symptoms initially showed as small, brown water-soaked spots on the leaf surface or margins, and enlarged to irregular, dark brown or black, necrotic lesions, with a lighter center and darker edge. At latter, lesions extended to most of the leaf surface causing leaf scorch or wilting. Almost 40% of the plants surveyed were infected. Symptomatic leaf samples were collected, and small pieces were taken at their disease-healthy junction, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) for incubation at 26 °C for 5 days in the dark. Fungal colonies with similar morphology were observed and, in total, 10 isolates were obtained from 10 plants. On PDA, colonies were initially white with fluffy hyphae, and later became light to dark gray, showing faint concentric rings. Conidia were hyaline, aseptate, cylindrical, and rounded at both ends, measuring 11.36 to 17.67 × 3.45 to 5.9 μm (n = 50). Appressoria were dark brown, ovate, globose, measuring 6.37 to 7.55 × 10.11 to 12.3 µm. These morphological characteristics were typical of Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of a representative isolate Cs-8-5-1 were amplified and sequenced using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF/GDR as described previously (Weir et al. 2012). These sequences were deposited in GenBank (accession nos. OM439575 for ITS, OM459820 for ACT, OM459821 for CHS-1, and OM459822 for GAPDH). BLASTn analysis showed 99.59 to 100 % identity to the corresponding sequences of C. siamense strains. A Maximum likelihood phylogenetic tree based on the concatenated ITS, ACT, CHS-1 and GAPDH sequences was generated by MEGA 6. It revealed that the Cs-8-5-1 was clustered with the C. siamense strain CBS 132456 with 98% bootstrap support. For pathogenicity test, conidia suspension (105 spores/ml) was prepared by harvesting conidia from 7-day-old cultures growing on PDA, and 10 uL was dropped onto leaves of potted D. alata plants (8 droplets per leaf). Leaves treated with sterile water were served as controls. All the inoculated plants were placed in humid chambers (with 90% humidity) at 26°C with a photoperiod of 12 h. The pathogenicity tests were performed twice, with each had three replicated plants. Seven days after inoculation, the inoculated leaves showed symptoms of brown necrosis resembling that observed in fields, however, the control leaves remained symptomless. The fungus was specifically re-isolated and identified by morphological and molecular methods, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on D. alata in China. Since this disease might seriously affect the photosynthesis of the plants, which can influence the yield, prevention and management strategies should be adopted to control this new disease. Identification of this pathogen will provide a foundation for the diagnosis and control of this disease.

First report of Colletotrichum fructicola causing anthracnose on peanut (Arachis hypogaea L.) in China.

Peanut (Arachis hypogaea L.) is an important cash crop and oil crop around the world. In August 2021, symptoms of leaf spot were found on nearly 50% of peanut plants in the peanut planting base of Xuzhou Academy of Agriculture Sciences, Jiangsu, China. Symptoms began as small, round or oval, dark brown spots on the leaf. As the spot expanded, the center of the spot became gray to light brown and the spot was covered with small black dots. Fifteen leaves with typical symptoms were randomly collected from fifteen plants in three fields about a kilometer apart from each other. Leaf pieces (5 × 5 mm) were cut from the junction part of diseased and healthy leaf tissue, sterilized with 75% ethanol for 30 s and 5% NaClO for 30 s, washed 3 times with sterile water, placed on full strength potato dextrose agar (PDA) and incubated at 28°C in darkness. Five days after incubation, 12 isolates were obtained. Fungal colonies were white to gray on the upper surface and orange to gray on the reverse side. Conidia were single-celled, cylindrical and colorless after maturation, and were 12 - 16.5 × 4.5 - 5.5 μm (n = 50) in size. Ascospores were one-celled, hyaline, with tapering ends and one or two large guttulates at the center, and measured 9.4 - 21.5 × 4.3 - 6.4 μm (n = 50). Based on morphological characteristics, the fungi were preliminarily identified as Colletotrichum fructicola (Prihastuti et al. 2009; Rojas et al. 2010). Single spore isolates were cultured on PDA medium and two representative strains (Y18-3 and Y23-4) were selected for DNA extraction. The internal transcribed spacer (ITS) rDNA region, partial actin gene (ACT), partial calmodulin gene (CAL), partial chitin synthase gene (CHS), partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and partial beta-tubulin 2 gene (TUB2) were amplified. The nucleotide sequences were submitted to Genbank (accession numbers of strain Y18-3: ITS: ON619598; ACT: ON638735; CAL: ON773430; CHS: ON773432; GAPDH: ON773436; TUB2: ON773434; accession numbers of strain Y23-4: ITS: ON620093; ACT: ON773438; CAL: ON773431; CHS: ON773433; GAPDH: ON773437; TUB2: ON773435). The phylogenetic tree was constructed using MEGA 7 based on the tandem of six genes (ITS-ACT-CAL-CHS-GAPDH-TUB2). The result showed that isolates Y18-3 and Y23-4 reside in the clade of C. fructicola species. To determine pathogenicity, conidial suspensions (107/mL) of isolate Y18-3 and Y23-4 were sprayed on ten 30-day-old healthy peanut seedlings per isolate. Five control plants were sprayed with sterile water. All plants were kept moist at 28°C in the dark (> 85% RH) for 48 h and then transferred to a moist chamber at 25°C with a 14-h photoperiod. After two weeks, typical anthracnose symptoms similar to those observed in the field appeared on leaves of inoculated plants, whereas controls remained asymptomatic. C. fructicola was re-isolated from symptomatic leaves but not from controls. Koch's postulates verified that C. fructicola was the pathogen of peanut anthracnose. C. fructicola is a well-known fungus causing anthracnose on many plant species worldwide. In recent years, new plant species infected by C. fructicola have been reported, like cherry, water hyacinth and Phoebe sheareri (Tang et al. 2021; Huang et al. 2021; Huang et al. 2022). To our knowledge, this is the first report of C. fructicola causing peanut anthracnose in China. Thus, it is recommended to pay close attention and take necessary prevention and control measures against potential spread of peanut anthracnose in China. .