First Report of Alternaria alternata Causing Leaf Spot on Coffea arabica in China.

Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 µm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.

Efficient production of butyric acid from lignocellulosic biomass by revealing the mechanisms of Clostridium tyrobutyricum tolerance to phenolic inhibitors.

Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain's tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.

Endophytic bacterial communities in wild rice (Oryza officinalis) and their plant growth-promoting effects on perennial rice.

Endophytic bacterial microbiomes of plants contribute to the physiological health of the host and its adaptive evolution and stress tolerance. Wild rice possesses enriched endophytic bacteria diversity, which is a potential resource for sustainable agriculture. Oryza officinalis is a unique perennial wild rice species in China with rich genetic resources. However, endophytic bacterial communities of this species and their plant growth-promoting (PGP) traits remain largely unknown. In this study, endophytic bacteria in the root, stem, and leaf tissues of O. officinalis were characterized using 16S rRNA gene Illumina sequencing. Culturable bacterial endophytes were also isolated from O. officinalis tissues and characterized for their PGP traits. The microbiome analysis showed a more complex structure and powerful function of the endophytic bacterial community in roots compared with those in other tissue compartments. Each compartment had its specific endophytic bacterial biomarkers, including Desulfomonile and Ruminiclostridium for roots; Lactobacillus, Acinetobacter, Cutibacterium and Dechloromonas for stems; and Stenotrophomonas, Chryseobacterium, Achromobacter and Methylobacterium for leaves. A total of 96 endophytic bacterial strains with PGP traits of phosphate solubilization, potassium release, nitrogen fixation, 1-aminocyclopropane-1-carboxylate (ACC) deaminase secretion, and siderophore or indole-3-acetic acid (IAA) production were isolated from O. officinalis. Among them, 11 strains identified as Enterobacter mori, E. ludwigii, E. cloacae, Bacillus amyloliquefaciens, B. siamensis, Pseudomonas rhodesiae and Kosakonia oryzae were selected for inoculation of perennial rice based on their IAA production traits. These strains showed promising PGP effects on perennial rice seedlings. They promoted plants to form a strong root system, stimulate biomass accumulation, and increase chlorophyll content and nitrogen uptake, which could fulfil the ecologically sustainable cultivation model of perennial rice. These results provide insights into the bacterial endosphere of O. officinalis and its application potential in perennial rice. There is the prospect of mining beneficial endophytic bacteria from wild rice species, which could rewild the microbiome of cultivated rice varieties and promote their growth.

First Report of Fusarium oxysporum Causing Leaf Wilt on Dinteranthus vanzylii (Green Stone Plant) in China.

Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.

Transcriptome analysis reveals reasons for the low tolerance of Clostridium tyrobutyricum to furan derivatives.

Lignocellulosic biomass is considered the most abundant and renewable feedstock for biobased butyric acid production. However, the furan derivatives (FAs, mainly furfural and 5-hydroxymethylfurfural) generated from the pretreatment of lignocellulose severely inhibit the growth of Clostridium tyrobutyricum, which is the best strain for producing butyric acid. The tolerance mechanism of C. tyrobutyricum to FAs has not been investigated thus far. Here, the response of C. tyrobutyricum ATCC 25755 to FA challenge was first evaluated by using comprehensive transcriptional analysis. The results indicated that the genes related to membrane transport, heat shock proteins, and transcriptional regulation were upregulated under FA stress. However, the expression of almost all genes encoding reductases was not changed, and only the ad gene CTK_RS02625 and the bud gene CTK_RS07810 showed a significant increase of ~ 1.05-fold. Then, the enzyme activity assays indicated that BUD could catalyze the reduction of FAs with relatively low activity and that AD could not participate in the conversion of FAs, indicating that the inability to rapidly convert FAs to their low-toxicity alcohols may be the main reason for the low FA tolerance of C. tyrobutyricum. This research provides insights into the development of FA-tolerant strains, thereby enhancing the bioconversion of lignocellulosic biomass to butyric acid. KEY POINTS: • The response of C. tyrobutyricum to FAs was evaluated for the first time. • Genes encoding membrane transporters and heat shock proteins were triggered by FAs. • A lack of effective FA reductases leads to low FA tolerance in C. tyrobutyricum.

First Report of Fusarium oxysporum Causing Stem Rot on Mammillaria humboldtii in China.

Mammillaria humboldtii found in Mexico is a short-globose ornamental cactus species of the Cactaceae family, which has gained increasing popularity in China. It is characterized by tuberculate stems, dimorphic areoles, small pink flowers and pitted seed cell walls. The populations of wild M. humboldtii are critically endangered and are now of international conservation concern. In July 2021, stem rot symptoms were observed on M. humboldtii in a commercial greenhouse located in Zhangzhou (117°39'44.0064″E, 24°28'3.7236″N), Fujian Province (southern China). The typical symptoms were water-soaking, rotting and wilting on the stem, eventually leading to necrosis of the plants within 20 to 30 days. The vascular system of infected stems and roots showed a reddish-brown discolouration. The disease affected approximately 10% of 1000 plants. Fungi were isolated from the diseased stems of 26 samples, which were chopped into small pieces (5 × 5 mm), surface-sterilized with 75% ethanol for 40 s, and placed onto potato dextrose agar (PDA). After seven days of dark culture at 28°C, morphologically similar fungal isolates with whitish aerial mycelium and purple pigment were observed. On carnation leaf agar (CLA), isolates produced sickle and slightly curved macroconidia with three to four septa, measuring 12.8 to 27.9 × 1.9 to 3.8 µm (n = 15), and unicellular, ovoid to elliptical microconidia measuring 3.8 to 7.7 × 1.4 to 2.5 µm (n = 30). Smooth walled chlamydospores were terminal or intercalary, single or in pairs, measuring 9.2 to 13.1 µm (n = 15) in diameter. For molecular identification, the internal transcribed spacer (ITS) region of rDNA (Schoch et al. 2012), translation elongation factor-1α (EF1-α) (Maryani et al. 2019) and gene coding endopolygalacturonase 1 (PG1) (Hirano et al. 2006) of the representative isolate FJMH7 were amplified, purified and sequenced. BLASTn analysis of the ITS, EF1-α and pg1 sequences (GenBank accession numbers: ON832660, ON843495 and ON843496) showed 100%, 99.70% and 98.96% identity with F. oxysporum (GenBank accession numbers: KX611626, OM801797 and KF437345), respectively. Phylogenetic analysis based on the the concatenated ITS and EF1-α sequences and pg1 genes placed isolate FJMH7 with F. oxysporum reference strains in the phylogenetic trees. Based on morphological identification and sequence analysis, this isolate was identified as F. oxysporum. For the pathogenicity assay, six 6-month-old healthy plants of Mammillaria humboldtii were inoculated by dipping roots in a conidial suspension (106 conidia/mL) of isolate FJMH7 cultured in Bilai's medium for three days. Six noninoculated plants treated with Bilai's medium served as a control. Plants were transplanted into pots filled with sterilized soils and placed in a glasshouse at 25°C. After 15 days, all the inoculated plants exhibited rot symptoms on stems, which were similar to those observed in the commercial greenhouses. All inoculated plants were dead 30 days after inoculation. Control plants did not show any symptoms. F. oxysporum was reisolated and confirmed based on morphology and sequencing. No fungi were reisolated from control plants. To fulfil Koch's postulates, the pathogenicity assay was repeated twice with the same results. To date, F. oxysporum isolates have been reported on golden barrel cactus (Echinocactus grusonii) (Polizzi et al. 2004), night-blooming cereus (Hylocereus undatus) (Wright et al. 2007), apple cactus (Cereus peruvianus monstruosus) (Garibaldi et al. 2011), Schlumbergera truncate (Lops et al. 2013), Astrophytum ornatum (Quezada-Salinas et al. 2017) and Nopalea cochenillifera (Santiago et al. 2018). To our knowledge, this is the first report of F. oxysporum on M. humboldtii in China, indicating that this pathogen could cause wilt and rot disease on different cactus hosts.

The Effects of Different Processing Methods on the Levels of Biogenic Amines in Zijuan Tea.

This study aimed to evaluate the effects of processing methods on the content of biogenic amines in Zijuan tea by using derivatization and hot trichloroacetic acid extraction with HPLC-UV. The results showed that the most abundant biogenic amine in the original leaves was butylamine, followed by ethylamine, methylamine, 1,7-diaminoheptane, histamine, tyramine, and 2-phenethylamine. However, during the process of producing green tea, white tea, and black tea, the content of ethylamine increased sharply, which directly led to their total contents of biogenic amines increasing by 184.4%, 169.3%, and 178.7% compared with that of the original leaves, respectively. Unexpectedly, the contents of methylamine, ethylamine, butylamine, and tyramine in dark tea were significantly reduced compared with those of the original leaves. Accordingly, the total content of biogenic amines in dark tea was only 161.19 µg/g, a reduction of 47.2% compared with that of the original leaves, indicating that the pile-fermentation process could significantly degrade the biogenic amines present in dark tea.

Butyric acid production from spent coffee grounds by engineered Clostridium tyrobutyricum overexpressing galactose catabolism genes.

Clostridium tyrobutyricum cannot utilize galactose, which is abundant in lignocellulose and red algae, as a carbon source for butyric acid production. Hence, when using galactose-rich coffee ground hydrolysate as the substrate, the fermentation performance of C. tyrobutyricum is poor. In this work, a recombinant strain, C. tyrobutyricum ATCC 25755/ketp, overexpressing galactose catabolism genes (galK, galE, galT, and galP) from Clostridium acetobutylicum ATCC 824 was constructed for the co-utilization of glucose and galactose. Batch fermentation in the bioreactor showed that ATCC 25755/ketp could efficiently utilize galactose without glucose-induced carbon catabolite repression and consume nearly 100% of the galactose present in the spent coffee ground hydrolysate. Correspondingly, the butyric acid concentration and productivity of ATCC 25755/ketp reached 34.3 g/L and 0.36 g/L·h, respectively, an increase of 78.6% and 56.5% compared with the wild-type strain, indicating its potential for butyric acid production from hydrolysates of inexpensive and galactose-rich biomass.

Comparative phosphoproteomic analysis of blast resistant and susceptible rice cultivars in response to salicylic acid.

BACKGROUND: Salicylic acid (SA) is a significant signaling molecule that induces rice resistance against pathogen invasion. Protein phosphorylation carries out an important regulatory function in plant defense responses, while the global phosphoproteome changes in rice response to SA-mediated defense response has not been reported. In this study, a comparative phosphoproteomic profiling was conducted by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis, with two near-isogenic rice cultivars after SA treatment. RESULTS: Thirty-seven phosphoprotein spots were differentially expressed after SA treatment, twenty-nine of which were identified by MALDI-TOF/TOF MS, belonging to nine functional categories. Phosphoproteins involved in photosynthesis, antioxidative enzymes, molecular chaperones were similarly expressed in the two cultivars, suggesting SA might alleviate decreases in plant photosynthesis, regulate the antioxidant defense activities, thus improving basal resistance response in both cultivars. Meanwhile, phosphoproteins related to defense, carbohydrate metabolism, protein synthesis and degradation were differentially expressed, suggesting phosphorylation regulation mediated by SA may coordinate complex cellular activities in the two cultivars. Furthermore, the phosphorylation sites of four identified phosphoproteins were verified by NanoLC-MS/MS, and phosphorylated regulation of three enzymes (cinnamoyl-CoA reductase, phosphoglycerate mutase and ascorbate peroxidase) was validated by activity determination. CONCLUSIONS: Our study suggested that phosphorylation regulation mediated by SA may contribute to the different resistance response of the two cultivars. To our knowledge, this is the first report to measure rice phosphoproteomic changes in response to SA, which provides new insights into molecular mechanisms of SA-induced rice defense.

Comparative Transcriptomic Analysis of Race 1 and Race 4 of Fusarium oxysporum f. sp. cubense Induced with Different Carbon Sources.

The fungal pathogen Fusarium oxysporum f. sp. cubense causes Fusarium wilt, one of the most destructive diseases in banana and plantain cultivars. Pathogenic race 1 attacks the "Gros Michel" banana cultivar, and race 4 is pathogenic to the Cavendish banana cultivar and those cultivars that are susceptible to Foc1. To understand the divergence in gene expression modules between the two races during degradation of the host cell wall, we performed RNA sequencing to compare the genome-wide transcriptional profiles of the two races grown in media containing banana cell wall, pectin, or glucose as the sole carbon source. Overall, the gene expression profiles of Foc1 and Foc4 in response to host cell wall or pectin appeared remarkably different. When grown with host cell wall, a much larger number of genes showed altered levels of expression in Foc4 in comparison with Foc1, including genes encoding carbohydrate-active enzymes (CAZymes) and other virulence-related genes. Additionally, the levels of gene expression were higher in Foc4 than in Foc1 when grown with host cell wall or pectin. Furthermore, a great majority of genes were differentially expressed in a variety-specific manner when induced by host cell wall or pectin. More specific CAZymes and other pathogenesis-related genes were expressed in Foc4 than in Foc1 when grown with host cell wall. The first transcriptome profiles obtained for Foc during degradation of the host cell wall may provide new insights into the mechanism of banana cell wall polysaccharide decomposition and the genetic basis of Foc host specificity.

[Role of dendritic cells in the pathogenesis of asthma in children].

UNLABELLED: Dendritic cells (DC) are very potent antigen-presenting cells (APC) with a unique ability to activate naive T cells to induce the differentiation of TH1/TH2. Monocytes can develop into DC in the presence of different cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. DCs are thought to play a key role in the initiation and maintenance of T cell immunity to inhaled antigens. While the density of DC within the bronchial mucosa is increased in asthma, there is little information currently available concerning the effects of DC in asthmatic children. OBJECTIVE: To investigate the role of dendritic cells in the pathogenesis of acute attack of asthma in children. METHODS: Thomas' method was adopted. The adherent precursors of DC were isolated from peripheral blood of asthmatic children in acute attack stage and healthy controls. The adherent cells were induced with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) to DC in vitro. The expression of the surface molecules CD80, CD86, HLA-DR etc. on the DC was examined by fluorescent activated cell sorter (FACS). And the ability to secret IL-10, IL-12 and their potentials to stimulate the proliferative reaction of DC inductive self T- lymphocyte were observed. RESULTS: The results showed that in asthmatic children's acute attack stage, self T- lymphocyte proliferative reaction induced by DC was remarkably increased compared with normal control subjects (P < 0.01). At the same time, the asthmatic children in acute attack stage had remarkably decreased the ability to secret IL-10 compared with normal control subjects (P < 0.01), while the ability to secret IL-12 remarkably decreased compared with normal control subjects (P < 0.01); meanwhile, the HLA-DR and co-stimulating factor CD86(B(7-2)) expressed by DCs remarkably increased in the asthmatic children in acute attack stage compared with normal control subjects (P < 0.01). CONCLUSION: DC possibly plays a vital role in the immunological mechanism of asthma by means of inducing the differentiation of TH1/TH2, that is DC may be the key factor in initiating the airway allergic reaction and the possible mechanism may involve interleukins (especially IL-10 and IL-12, etc.) secreted by DCs.