Screening of Antagonistic Bacillus against Brown Rot in Dendrocalamus latiflorus and Preparation of Applying Bacterial Suspension.

The aim of this study was to isolate biocontrol bacteria that could antagonize brown rot of Dendrocalamus latiflorus, optimize the culture conditions, and develop an effective biocontrol preparation for brown rot of D. latiflorus. This study isolated a bacterium with an antagonistic effect on bamboo brown rot from healthy D. latiflorus rhizosphere soil. Morphology, molecular biology, and physiological biochemistry methods identified it as Bacillus siamensis. The following culturing media and conditions improved the inhibition effect of B. siamensis: the best culturing media were 2% sucrose, 1.5% yeast extract, and 0.7% potassium chloride; the optimal culturing time, temperature, pH, and inoculation amount were 48 h, 30℃, 6, and 20%. The optimum formula of the applying bacterial suspension was 14% sodium dodecyl benzene sulfonate emulsifier, 4% Na2HPO4·2H2O, 0.3% hydroxypropyl methylcellulose thickener, and 20% B. siamensis. The pot experiment results showed the control effect of applying bacterial suspension, diluted 1,000 times is still better than that of 24% fenbuconazole suspension. The applying bacterial suspension enables reliable control of brown rot in D. latiflorus.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/ß-Sandy-R for the ß-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

Functional Identification of Arthrinium phaeospermum Effectors Related to Bambusa pervariabilis × Dendrocalamopsis grandis Shoot Blight.

The shoot blight of Bambusa pervariabilis × Dendrocalamopsis grandis caused by Arthrinium phaeospermum made bamboo die in a large area, resulting in serious ecological and economic losses. Dual RNA-seq was used to sequence and analyze the transcriptome data of A. phaeospermum and B. pervariabilis × D. grandis in the four periods after the pathogen infected the host and to screen the candidate effectors of the pathogen related to the infection. After the identification of the effectors by the tobacco transient expression system, the functions of these effectors were verified by gene knockout. Fifty-three differentially expressed candidate effectors were obtained by differential gene expression analysis and effector prediction. Among them, the effectors ApCE12 and ApCE22 can cause programmed cell death in tobacco. The disease index of B. pervariabilis × D. grandis inoculated with mutant ΔApCE12 and mutant ΔApCE22 strains were 52.5% and 47.5%, respectively, which was significantly lower than that of the wild-type strains (80%), the ApCE12 complementary strain (77.5%), and the ApCE22 complementary strain (75%). The tolerance of the mutant ΔApCE12 and mutant ΔApCE22 strains to H2O2 and NaCl stress was significantly lower than that of the wild-type strain and the ApCE12 complementary and ApCE22 complementary strains, but there was no difference in their tolerance to Congo red. Therefore, this study shows that the effectors ApCE12 and ApCE22 play an important role in A. phaeospermum virulence and response to H2O2 and NaCl stress.

Molecular Mechanisms of Phenylpropane-Synthesis-Related Genes Regulating the Shoot Blight Resistance of Bambusa pervariabilis × Dendrocalamopsis grandis.

Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight caused by Arthrinium phaeospermum is a fungal disease that has affected a large area in China in recent years. However, it is not clear which genes are responsible for the disease resistance of B. pervariabilis × D. grandis. Based on the analysis of transcriptome and proteome data, two genes, CCoAOMT2 and CAD5, which may be involved in disease resistance, were screened. Two gene expression-interfering varieties, COF RNAi and CAD RNAi were successfully obtained using RNAi technology. Quantitative real-time fluorescence (qRT-PCR) results showed that CCoAOMT2 gene, CAD5 gene and seven related genes expression was down-regulated in the transformed varieties. After inoculating pathogen spore suspension, the incidence and disease index of cof-RNAi and cad-RNAi transformed plants increased significantly. At the same time, it was found that the content of total lignin and flavonoids in the two transformed varieties were significantly lower than that of the wild-type. The subcellular localization results showed that both CCoAOMT2 and CAD5 were localized in the nucleus and cytoplasm. The above results confirm that the CCoAOMT2 and CAD5 genes are involved in the resistance of B. pervariabilis × D.grandis to shoot blight through regulating the synthesis of lignin and flavonoids.

Differential transcriptome analysis and identification of genes related to resistance to blight in three varieties of Bambusa pervariabilis × Dendrocalamopsis grandis.

BACKGROUND: Bambusa pervariabilis × Dendrocalamopsis grandis is a fast-growing bamboo that is widely introduced in southern China and has great economic and ecological benefits. In recent years, a blight of B. pervariabilis × D. grandis caused by Arthrinium phaeospermum has led to much branch damage and even death of entire bamboo forests. METHODS: To screen for resistance genes in B. pervariabilis × D. grandis, transcriptome sequencing technology was used to compare the gene expression profiles of different varieties of B. pervariabilis × D. grandis with variable resistance and the same varieties under different treatments. The Clusters of Orthologous Groups of Proteins (COG) database; the Gene Ontology (GO) database; and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to annotate and analyse the differentially expressed genes. RESULTS: A total of 26,157 and 11,648 differentially expressed genes were obtained in the different varieties after inoculation with A. phaeospermum and the same varieties after inoculation A. phaeospermum or sterile water, respectively. There were 23 co-upregulated DGEs and 143 co-downregulated DEGs in #3 and #8, #6 and #8, #6 and #3. There were 50 co-upregulated DGEs and 24 co-downregulated DEGs in the same varieties after inoculation A. phaeospermum or sterile water. The results showed that many genes involved in cell wall composition synthesis, redox reactions and signal transduction were significantly different after pathogen infection. Twenty-one candidate genes for blight resistance, such as pme53, cad5, pod, gdsl-ll and Myb4l, were found. The qRT-PCR results were consistent with the sequencing results, verifying their authenticity. These results provide a foundation for the further exploration of resistance genes and their functions.

Brown Culm Rot of Dendrocalamus latiflorus Munro Caused by Diaporthe guangxiensis in Sichuan Province, China.

Dendrocalamus latiflorus Munro, the most widely cultivated bamboo species in southern China, has high ornamental value used in gardens, while culms are also used for buildings and as fibers and edibles (Gao et al. 2011). In June 2020, brown culm rot of bamboo was observed in Yibin city, Sichuan Province, in an area of approximately 1000 hectares. Disease incidence was approximately 60%, of which 30% of the plants had died. At the end of June, the lesions expanded but did not surround the base of the culm. From the end of June to the beginning of September, the lesions expanded upward and formed a streak, of which the color gradually deepened to purple-brown and black-brown. At the same time, the disease spots at the base of the culm also expanded horizontally. After the spots surrounded the base of the culm, the diseased bamboo died. Ten culms showing typical symptoms were collected and cut into 5×5 mm pieces at the junction of infected and healthy tissues. The tissues were sterilized for 1 to 2 min in 3% sodium hypochlorite, decontaminated in 75% alcohol for 3 to 5 min, placed on modified potato glucose agar (PDA) with streptomycin sulfate (50 µg/ml), and incubated at 26°C. Two isolates were obtained by the single-spore method (Sivan et al. 1992). The isolates both produced white round colonies similar to Diaporthe guangxiensis and two types of conidia: one was α type (5.5 to 8.2×1.0 to 2.8 µm, n=30), colourless, single-celled, undivided, and oval, containing two oil droplets; and ß type (21.1 to 30.2×0.8 to 1.4 µm, n=30), colourless, single celled and hook shaped. Genomic DNA was extracted from the two isolates by using a fungal genomic DNA extraction kit (Solarbio, Beijing). The products were amplified by polymerase chain reaction (PCR) with primers for the internal transcribed spacer 1 (ITS) region (White et al. 1990), calmodulin (CAL) gene (Carbone and Kohn 1999), translation elongation factor 1-alpha (TEF) gene (Glass and Donaldson 1995) and beta-tubulin (TUB) gene (Soares et al. 2018). The amplified products were sequenced and blasted in GenBank (accession numbers MW380383, MW431318, MW431317 and MW431316 for ITS, CAL, TEF, and TUB, respectively). The ITS, CAL, TEF, and TUB sequences showed 100%, 99.33%, 100%, and 99.80% identity to D. guangxiensis JZB320094 (accession numbers MK335772.1, MK736727.1, MK523566.1, MK500168.1 in GenBank), respectively. To evaluate the pathogenicity of the isolates, five plants were each inoculated with two isolates. The cortex of potted bamboo were injured locally with sterilized needle, and the bamboo culms were inoculated with 100 µl of conidial suspension (105 cfu/ml). The surface of the inoculation wound was covered with gauze soaked with sterilized water. Five plants inoculated with sterile water were used as controls. The treated plants were maintained in a greenhouse at a temperature of 22 to 29°C and relative humidity of 70 to 80%. One month later, of all inoculated plants showed similar symptoms as those observed in the field. D. guangxiensis was re-isolated from all inoculated plants. The pathogenicity test was repeated three times with similar results. This is the first report of D. guangxiensis causing brown culm rot of D. latiflorus in China. These results will facilitate an enhanced understanding of factors affecting bamboo and the design of effective management strategies of the pathogenic species on bamboo and thus to develop corresponding control measures.

Leaf Spot Disease Caused by Pestalotiopsis kenyana on Zanthoxylum schinifolium in Sichuan Province, China.

Zanthoxylum schinifolium Sieb. et Zucc, a species of prickly ash, is one of the main economic plants in China and mainly grown in Southwest China. The planting area of Z. schinifolium accounts for more than 70% of the total area of prickly ash, and one of the largest plantings of Z. schinifolium is located in Jianyang City (Sichuan) with the area of 6.67 km2. Since 2018, Z. schinifolium, located in Jianyang City, have developed leaf spot disease, with approximately 50% showing disease symptoms. At the beginning of the occurrence, yellow-brown lesions formed on the leaves; in the later stages, the area of the lesions expanded. At the severe stage, multiple lesions merged into one large, dead spot, and the plants failed to blossom and bear fruit. The samples were collected from typical symptoms of Z. schinifolium leaves in Jianyang City. A total of 20 leaf samples were collected from 5 Z. schinifolium plants (4 leaves per plant), and were cut into small pieces of 2 × 2mm at the junction of infected and healthy tissues. These tissues were surface-disinfested for 30 s in 3% sodium hypochlorite and the for 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in a dark incubator at 25°C. Morphological observation was performed on 18 recovered isolates, 15 of which were described as Pestalotiopsis sp. The colonies were incubated on PDA at 25°C for 7 days and reached a diameter of 80-90 mm. The colonies were white with undulating edges and were similar in colors on the reverse side. After colony culture at 25°C for 10 days, gregarious black conidiomata were scattered on the mycelial mats. The conidia and appendages of the samples were measured by Leica Application Suite X 3.4.1.17822 (20 conidia per isolate), and the sizes of which were consistent with the description from Maharachchikumbura et al. Based on morphological observations, the isolates were identified as Pestalotiopsis kenyana Maharachch., K.D. Hyde & Crous. PCR was performed with primers ITS1/ITS4 for the ITS region, primers D1/D2 for the large subunit ribosomal RNA gene (LSU), primers 5f2/7cr for the RNA polymerase II second largest subunit (RPB2), primers Bt2a/Bt2b for the ß-tubulin gene (TUB), and primers EF1-526F/EF2-567R for the translation elongation factor 1-alpha gene (TEF). The Sanger-sequenced PCR products were sequenced and blasted in GenBank, and the sequences showed that ITS: 99.17% (594 out of 599 bp), LSU: 100% (909 out of 909 bp), RPB2: 99.17% (832 out of 832 bp), TUB: 100% (774 out of 774 bp), TEF: 100% (485 out of 485 bp) with the type specimen of P. kenyana CBS 442.67 (ITS: GenBank accession NR147549.1, LSU: MH870724.1, PRB2: MH554958.1, TUB: KM199395.1, TEF: KM199502.1). Representative sequences were deposited in GenBank (ITS: MT509798; LSU: MT509800; RPB2: MT522448; TUB: MT522450; TEF: MT522449). To fulfill Koch's postulates, leaves on fifteen one-year-old healthy potted Z. schinifolium plants were sterilized by 75% ethanol cotton balls, and were rinsed by sterile water for three times. Then each leaf was punctured with sterile needles for two wounds (five leaves per plant). The wounds were inoculated by placing 8 mm mycelial plugs obtained from the periphery of 7-day-old single-spore cultures. An equal number of plants were wounded with the same method, and were respectively inoculated with sterile water and PDA plugs without mycelium as controls. All plants were placed in a growth chamber at 25°C under 90% relative humidity. After 7 days, all mycelial-inoculated leaves of the plants showed symptoms identical to those described above, whereas the control plants remained symptom free. P. kenyana was re-isolated from the infected leaves and confirmed to be the same as the inoculated pathogen through analyses of morphological characteristics and molecular techniques. The pathogenicity test was repeated three times with similar results. To our knowledge, this is the first report of P. kenyana as a causal agent of leaf spot disease on Z. schinifolium in China. These findings will aid the development of better preventive measures in accordance with the emergence of this new pathogen.

First Report of Fusarium fujikuroi Causing Black Stem Rot of Zanthoxylum bungeanum in China.

Chinese prickly ash (Zanthoxylum bungeanum Maxim.), native to China, is an important tree species for soil and water conservation, barren mountain afforestation, and garden greening. Its fruit is commonly used for seasoning and medicine. In August 2016, black stem rot of Z. bungeanum was first observed in Hanyuan County, Ya'an City. In June 2019, the symptoms were observed on > 60% of 10,000 plants in Hanyuan County. At its early stage, the bark was wet and rotten, slightly concave, and accompanied by gummosis. The lesions were dark brown and long oval, peeling off the rotten bark covered with white hyphae. At the later stage, the lesions shrunk and cracked, with many orange-red particles (conidia) and dense black particles (ascospores). Larger lesions often caused large-scale bark necrosis. After the lesions girdled the trunk, the plants rapidly died. A total of 36 isolates were isolated from 320 infested tissue fragments (5 × 5 mm) that were surface sterilized for 60 s in 3% sodium hypochlorite, and 60 s in 75% ethanol, rinsed three times in sterilized water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in the dark at 25°C. Among them, 28 exhibited morphological characteristics described as Fusarium fujikuroi Nirenberg. On PDA, the fungus produced white to grey orange, circular colonies. On carnation leaf agar (CLA), the microconidia were club shaped with a flattened base and 0 to 1 septum, 5 to 15 × 2 to 4 µm in size, whereas macroconidia were relatively slender, medium length with no significant curvature, and had a tapered apical cell and a poorly developed, notched basal cell, 3 to 5 septa, 20 to 50 × 3 to 5 µm in size. For molecular identification, DNA was extracted from a representative isolate using a fungus genomic DNA extraction kit (Solarbio, Beijing). PCRs were performed with primers EF1f/EF2r for translation elongation factor 1α (EF-1α) region, and primers 5f2/7cr for RNA polymerase II genes (RPB2) region. PCRs were amplified and their products (GenBank accession nos. MT448248 and MT448247) were sequenced and blasted, showing 99 to 100% sequence homology with known F. fujikuroi isolates (GenBank accession nos. MN102101.1 and MN193888.1). To conduct pathogenicity test, twigs of 42 three-year-old potted Z. bungeanum plants were superficially wounded with a needle on the cortex, and each twig was inoculated by dropping a 100 µl conidial suspension (1×106 conidia/ml) of the fungus onto its wound surface. The inoculated areas were bandaged with gauze moistened with sterilized water. The wounded wigs that were inoculated with sterilized water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 22 to 29°C and RH from 70% to 80%. One month later, 100% of inoculated plants showed the symptoms similar to those observed in the field, with the size of lesions ranging from 0.5 to 1.0 cm2. The fungus was re-isolated from the branch cortexes and confirmed morphologically and molecularly. To our knowledge, this is the first report of F. fujikuroi as a causal agent of black stem rot disease on Z. bungeanum in China. These results will help correctly identify this disease and develop proper strategies to manage the disease.

First Report of Arthrinium kogelbergense Causing Blight Disease of Bambusa intermedia in Sichuan Province, China.

Bambusa intermedia Hsueh et Yi, a species in the Gramineae family, is mainly distributed in southern China and is commonly found in the Sichuan, Yunnan, and Guangdong Provinces and in the Guangxi Autonomous Region. It is economically significant as a building material, a food source, and for applications in various other raw products. In July 2017, a blight disease was found on B. intermedia stems, affecting approximately 40% of 9600 plants in the Changning and Jiangan counties of Sichuan Province, China. In the early stages of the disease, leaves at the top of the plants withered and yellowed, eventually falling off. Subsequently, the stems and upper branches discolored and were dead to varying degrees. To isolate the causal fungus, 100 samples from B. intermedia culms and branches were collected from symptomatic plants in Changning and Jiangan counties. Small sections (4 to 5 mm2) were surface-sterilized for 30 s in 3% sodium hypochlorite and 60 s in 75% ethanol. The samples were then rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml, Solarbio, Beijing), and incubated in a dark incubator at 25 ± 1°C for 6 days. A total of 120 isolates were obtained, of which 89 isolates had similar colony morphology and conidial measurements. Colony characteristics of the fungus on the PDA surface were dirty white, reverse dirty white to pale luteous, colonies flat, floccose, with moderate aerial mycelium. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were erect, septate, pale brown, smooth, and reduced to conidiogenous cells. Conidiogenous cells were pale brown, smooth, and ampulliform. Conidia were 5.69-8.03 × 4.32-7.30 µm (30 conidia per isolate, Leica Application Suite X 3.4.1.17822) single celled, brown to black, smooth, and globose to ellipsoid shape, mostly globose in surface view and lenticular in side view with an equatorial ring. Based on morphological characteristics, it was identified as an Arthrinium species (Crous and Groenwald 2013). PCRs were performed with primers ITS4/ITS5 for the ITS region (White et al. 1990), primers T1/Bt2b for the ß-tubulin gene (TUB) (O'Donnell and Cigelnik 1997, Glass and Donaldson 1995) , primers 5.8sR/LR5 (Vilgalys and Hester 1990) and LR0R/TW13 (Hamayun et al. 2009) for 28S large-subunit (LSU) rDNA. Newly generated representative sequences were deposited in GenBank, ITS sequence (GenBank No. MT415395), TUB sequence (MT415365), and LSU sequence (MT415396, MT415398). Comparison to ex-type isolate sequences showed strong homology with Arthrinium kogelbergense Crous (CBS 113333, Crous and Groenwald 2013). ITS: 99.65% identity to Accession NR_120272; TUB: 99.75% identity to Accession KF144984; LSU: 99.60% and 100.00% identity to Accession NG_042779. To confirm its pathogenicity and to fulfill Koch's postulates, 20 one-year-old B. intermedia plants were wounded with a sterile needle at a depth of 1 mm in the stems and twigs, and were inoculated with a 5-mm-diameter disk of PDA that was colonized by the isolates (2 plants per isolate in each inoculation experiment, 10 repetitions) (Li et al. 2016). Ten control plants were treated similarly except that they were mock inoculated with PDA plugs without the fungus. All plants were kept at 25-28°C and covered with plastic bags to maintain high relative humidity (90-95%) on a 12-h light/dark incubation. Thirty days later, the inoculated plants showed the same symptoms observed originally, and the controls remained healthy. The same fungus was reisolated from the infected stems and twigs and showed similar morphological characteristics and molecular traits. To our knowledge, this is the first report of A. kogelbergense as a causal agent of blight disease on B. intermedia in Sichuan Province, China.