Comparative genomic analysis of Bacillus atrophaeus HAB-5 reveals genes associated with antimicrobial and plant growth-promoting activities.

Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

Rapid and Sensitive Detection of Verticillium dahliae from Soil Using LAMP-CRISPR/Cas12a Technology.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/µL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method's user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/µL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

Comparative Mitogenomics Analysis Revealed Evolutionary Divergence among Neopestalotiopsis Species Complex (Fungi: Xylariales).

The genus Neopestalotiopsis consists of obligate parasites that cause ring spot, scab, and leaf blight diseases in higher plant species. We assembled the three complete mitogenomes for the guava fruit ring spot pathogen, Neopestalotiopsis cubana. The mitogenomes are circular, with sizes of 38,666 bp, 33,846 bp, and 32,593 bp. The comparative analyses with Pestalotiopsis fici showed that N. cubana differs greatly from it in the length of the mitogenomes and the number of introns. Moreover, they showed significant differences in the gene content and tRNAs. The two genera showed little difference in gene skewness and codon preference for core protein-coding genes (PCGs). We compared gene sequencing in the mitogenomes of the order Xylariales and found large-scale gene rearrangement events, such as gene translocations and the duplication of tRNAs. N. cubana shows a unique evolutionary position in the phylum Ascomycota constructed in phylogenetic analyses. We also found a more concentrated distribution of evolutionary pressures on the PCGs of Neopestalotiopsis in the phylum Ascomycota and that they are under little selective pressure compared to other species and are subjected to purifying selection. This study explores the evolutionary dynamics of the mitogenomes of Neopestalotiopsis and provides important support for genetic and taxonomic studies.

Combined toxicity of trifloxystrobin and fluopyram to zebrafish embryos and the effect on bone development.

Trifloxystrobin (TRI) is a methacrylate fungicide, and fluopyram (FLU) is a new pyridylethylbenzamide fungicide and nematicide. Both are often detected in water bodies and may be highly toxic to many aquatic organisms. Unfortunately, the aquatic biological risks of single FLU or a mixture of trifloxystrobin and fluopyram have not been reported. In this study, zebrafish was selected as the test organism to investigate the combined toxicity of trifloxystrobin and fluopyram to zebrafish. After zebrafish embryos exposed to three pesticide solutions, Alcian-blue staining, Alizarin-red staining and quantitative PCR (qPCR) were performed. The results indicated that 96h-LC50 of TRI was 0.159 mg·L-1 to zebrafish embryo, which was highly toxic. The 96h-LC50 of FLU to zebrafish embryos was 4.375 mg·L-1, being moderately toxic. The joint toxicity to zebrafish embryos(FLU at 96h-LC50 and TRI at 96h-LC50 in a 1:1 weight ratio to form a series of concentration treatment groups) was antagonistic. Both trifloxystrobin and fluopyram also inhibited the skeletal development of zebrafish and showed to be antagonistic. The results of qPCR indicated upregulations of different genes upon three different treatments. TRI mainly induced Smads up-expression, which may affect the BMP-smads pathway. FLU mainly induced an up-expression of extracellular BMP ligands and type I receptor (Bmpr-1a), which may affect the BMP ligand receptor pathway. The 1:1 mixture (weight ratio) of trifloxystrobin and fluopyram induced a reduction of the genes of extracellular BMP ligand (Smads) and type I receptor (Bmpr1ba), which may down-regulate BMP signaling and thus attenuating cartilage hyperproliferation, hypertrophy and mineralization. The results warren an interest in further studying the effect of the two fungicides in a mixture on zebrafish.

Toxic effects of the insecticide tolfenpyrad on zebrafish embryos: Cardiac toxicity and mitochondrial damage.

Tolfenpyrad, a highly effective and broad-spectrum insecticide and acaricide extensively utilized in agriculture, presents a potential hazard to nontarget organisms. This study was designed to explore the toxic mechanisms of tolfenpyrad on zebrafish embryos. Between 24 and 96 h after exposure of the fertilized embryos to tolfenpyrad at concentrations ranging from 0.001 to 0.016 mg/L (96 h-LC50 = 0.017 mg/L), lethal effects were apparent, accompanied with notable anomalies including pericardial edema, increased pericardial area, diminished heart rate, and an elongated distance between the venous sinus and the arterial bulb. Tolfenpyrad elicited noteworthy alterations in the expression of genes pertinent to cardiac development and apoptosis, with the most pronounced changes observed in the cardiac development-related genes of bone morphogenetic protein 2b (bmp2b) and p53 upregulated modulator of apoptosis (puma). The findings underscore that tolfenpyrad induces severe cardiac toxicity and mitochondrial damage in zebrafish embryos. This data is imperative for a comprehensive assessment of tolfenpyrad risks to aquatic ecosystems, particularly considering the limited knowledge regarding its detrimental impact on aquatic vertebrates.

WRR4B contributes to a broad-spectrum disease resistance against powdery mildew in Arabidopsis.

Oidium heveae HN1106, a powdery mildew (PM) that infects rubber trees, has been found to trigger disease resistance in Arabidopsis thaliana through ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-, PHYTOALEXIN DEFICIENT 4 (PAD4)- and salicylic acid (SA)-mediated signalling pathways. In this study, a typical TOLL-INTERLEUKIN 1 RECEPTOR, NUCLEOTIDE-BINDING, LEUCINE-RICH REPEAT (TIR-NB-LRR)-encoding gene, WHITE RUST RESISTANCE 4 (WRR4B), was identified to be required for the resistance against O. heveae in Arabidopsis. The expression of WRR4B was upregulated by O. heveae inoculation, and WRR4B positively regulated the expression of genes involved in SA biosynthesis, such as EDS1, PAD4, ICS1 (ISOCHORISMATE SYNTHASE 1), SARD1 (SYSTEMIC-ACQUIRED RESISTANCE DEFICIENT 1) and CBP60g (CALMODULIN-BINDING PROTEIN 60 G). Furthermore, WRR4B triggered self-amplification, suggesting that WRR4B mediated plant resistance through taking part in the SA-based positive feedback loop. In addition, WRR4B induced an EDS1-dependent hypersensitive response in Nicotiana benthamiana and contributed to disease resistance against three other PM species: Podosphaera xanthii, Erysiphe quercicola and Erysiphe neolycopersici, indicating that WRR4B is a broad-spectrum disease resistance gene against PMs.

The fatty acid 2-hydroxylase CsSCS7 is a key hyphal growth factor and potential control target in Colletotrichum siamense.

SCS7 is a fatty acid 2-hydroxylase required for the synthesis of inositol phosphorylceramide but is not essential for normal growth in Saccharomyces cerevisiae. Here, we demonstrate that the Colletotrichum siamense SCS7 homolog CsSCS7 plays a key role in hyphal growth. The CsSCS7 deletion mutant showed strong hyphal growth inhibition, small conidia, and marginally reduced sporulation and also resulted in a sharp reduction in the full virulence and increasing the fungicide sensitivity. The three protein domains (a cytochrome b5 domain, a transmembrane domain, and a hydroxylase domain) are important to CsSCS7 protein function in hyphal growth. The fatty acid assay results revealed that the CsSCS7 gene is important for balancing the contents of multiple mid-long- and short-chain fatty acids. Additionally, the retarded growth and virulence of C. siamense ΔCsSCS7 can be recovered partly by the reintroduction of homologous sequences from Magnaporthe oryzae and Fusarium graminearum but not SCS7 of S. cerevisiae. In addition, the spraying of C. siamense with naked CsSCS7-double-stranded RNA (dsRNAs), which leads to RNAi, increases the inhibition of hyphal growth and slightly decreases disease lesions. Then, we used nano material Mg-Al-layered double hydroxide as carriers to deliver dsRNA, which significantly enhanced the control effect of dsRNA, and the lesion area was obviously reduced. These data indicated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.IMPORTANCECsSCS7, which is homologous to yeast fatty acid 2-hydroxylase SCS7, was confirmed to play a key role in the hyphal growth of Colletotrichum siamense and affect its virulence. The CsSCS7 gene is involved in the synthesis and metabolism of fatty acids. Homologs from the filamentous fungi Magnaporthe oryzae and Fusarium graminearum can recover the retarded growth and virulence of C. siamense ΔCsSCS7. The spraying of double-stranded RNAs targeting CsSCS7 can inhibit hyphal growth and reduce the disease lesion area to some extent. After using nano material Mg-Al layered double hydroxide as carrier, the inhibition rates were significantly increased. We demonstrated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.

The Plasma Membrane H+ ATPase CsPMA2 Regulates Lipid Droplet Formation, Appressorial Development and Virulence in Colletotrichum siamense.

Plasma membrane H+-ATPases (PMAs) play an important role in the pathogenicity of pathogenic fungi. Lipid droplets are important storage sites for neutral lipids in fungal conidia and hyphae and can be used by plant pathogenic fungi for infection. However, the relationship between plasma membrane H+-ATPase, lipid droplets and virulence remains unclear. Here, we characterized a plasma membrane H+-ATPase, CsPMA2, that plays a key role in lipid droplet formation, appresorial development and virulence in C. siamense. Deletion of CsPMA2 impaired C. siamense conidial size, conidial germination, appressorial development and virulence but did not affect hyphal growth. ΔCsPMA2 increased the sensitivity of C. siamense to phytic acid and oxalic acid. CsPMA2 was localized to lipids on the plasma membrane and intracellular membrane. Deletion of CsPMA2 significantly inhibited the accumulation of lipid droplets and significantly affected the contents of some species of lipids, including 12 species with decreased lipid contents and 3 species with increased lipid contents. Furthermore, low pH can inhibit CsPMA2 expression and lipid droplet accumulation. Overall, our data revealed that the plasma membrane H+-ATPase CsPMA2 is involved in the regulation of lipid droplet formation and affects appressorial development and virulence in C. siamense.

Screening of compound-formulated Bacillus and its effect on plant growth promotion.

Bacillus bacteria can produce abundant secondary metabolites that are useful for biocontrol, especially in maintaining plant root microecology, and for plant protection. In this study, we determine the indicators of six Bacillus strains for colonization, promotion of plant growth, antimicrobial activity, and other aspects, with the aim of obtaining a compound bacteriological agent to construct a beneficial Bacillus microbial community in plant roots. We found that there was no significant difference in the growth curves of the six Bacillus strains over 12 h. However, strain HN-2 was found to have the strongest swimming ability and the highest bacteriostatic effect of n-butanol extract on the blight-causing bacteria Xanthomonas oryzae pv. oryzicola. The hemolytic circle produced by the n-butanol extract of strain FZB42 was the largest (8.67 ± 0.13 mm) and had the greatest bacteriostatic effect on the fungal pathogen Colletotrichum gloeosporioides, with a bacteriostatic circle diameter of 21.74 ± 0.40 mm. Strains HN-2 and FZB42 can rapidly form biofilms. Time-of-flight mass spectrometry and hemolytic plate tests showed that strains HN-2 and FZB42 may have significantly different activities because of their ability to produce large quantities of lipopeptides (i.e., surfactin, iturin, and fengycin). Different growth-promoting experiments revealed that the strains FZB42, HN-2, HAB-2, and HAB-5 had better growth-promoting potential than the control, and therefore these four strains were compounded in an equal ratio and used to treat pepper seedlings through root irrigation. We found an increase in the stem thickness (13%), leaf dry weight (14%), leaf number (26%), and chlorophyll content (41%) of pepper seedlings treated with the composite-formulated bacterial solution compared to the optimal single-bacterial solution treatment. Furthermore, several of these indicators increased by an average of 30% in the composite solution-treated pepper seedlings compared with the control water treatment group. In conclusion, the composite solution obtained by compounding strains FZB42 (OD600 = 1.2), HN-2 (OD600 = 0.9), HAB-2 (OD600 = 0.9), and HAB-5 (OD600 = 1.2) in equal parts highlights the advantages of a single bacterial solution, which includes achieving good growth promotion and antagonistic effects against pathogenic bacteria. The promotion of this compound-formulated Bacillus can reduce the application of chemical pesticides and fertilizers; promote plant growth and development; avoid the imbalances of soil microbial communities and thus reduce the risk of plant disease; and provide an experimental basis for the production and application of various types of biological control preparations in the future.

Mitochondrial characteristics of the powdery mildew genus Erysiphe revealed an extraordinary evolution in protein-coding genes.

The genus Erysiphe was an obligate parasite causing powdery mildew disease on a wide range of higher plants. However, the knowledge of their mitogenome architecture for lifestyle adaptability was scarce. Here, we assembled the first complete mitogenome (190,559 bp in size) for rubber tree powdery mildew pathogen Erysiphe quercicola. Comparable analysis of the Erysiphe mitogenomes exhibited conserved gene content, genome organization and codon usage bias, but extensive dynamic intron gain/loss events were presented between Erysiphe species. The phylogeny of the Ascomycota species constructed in the phylogenetic analysis showed genetic divergences of the Erysiphe species. Compared with other distant saprophytic and plant pathogenic fungi, Erysiphe had a flat distribution of evolutionary pressures on fungal standard protein-coding genes (PCGs). The Erysiphe PCGs had the highest mean selection pressure. In particular, Erysiphe's cox1, nad1, cob and rps3 genes had the most elevated selection pressures among corresponding PCGs across fungal genera. Altogether, the investigations provided a novel insight into the potential evolutionary pattern of the genus Erysiphe to adapt obligate biotrophic lifestyle and promoted the understanding of the high plasticity and population evolution of fungal mitogenomes.

The Rubber Tree (Heveae brasiliensis) MLO Protein HbMLO12 Promotes Plant Susceptibility to Sustain Infection by a Powdery Mildew Fungus.

Powdery mildew severely affects several important crops and cash plants. Disruption of mildew resistance locus O (MLO) genes elevates resistance against powdery mildew in several plants. However, whether rubber tree (Heveae brasiliensis) MLO proteins are linked to susceptibility remains unknown, owing to technical limitations in the genetic manipulation of this woody plant. A previous study showed that the H. brasiliensis MLO-like protein HbMLO12 demonstrates high amino acid sequence similarity with the known Arabidopsis MLO protein AtMLO12. In this study, we investigated whether HbMLO12 regulates susceptibility to powdery mildew. H. brasiliensis leaves take up exogenously synthesized double-stranded RNAs (dsRNAs), and foliar application of dsRNA homologous to HbMLO12 gene specifically induces HbMLO12 silencing in H. brasiliensis leaf tissues. Notably, HbMLO12 silencing inhibited fungal infection and elevated the immune response during interaction with the rubber tree powdery mildew fungus. Furthermore, the heterologous expression of HbMLO12 suppressed bacterial flg22- and fungal chitin-induced immune responses and enhanced bacterial infection in Arabidopsis. Our study provides evidence that HbMLO12 contributes to susceptibility to powdery mildew. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of conidial autofluorescence in powdery mildew.

Autofluorescence is produced by endogenous fluorophores, such as NAD(P)H, lipofuscin, melanin, and riboflavin, indicating the accumulation of substances and the state of energy metabolism in organisms. As an obligate parasite, powdery mildew is wildly spread by air and parasitic crops. However, most identification studies have been based on morphology and molecular biology which were far too time- and labor-consuming, thus lacking characteristic, simple, and effective means. Using microscopy under the blue and cyan channels, we elaborated visible conidial autofluorescence in three powdery mildew species, Erysiphe quercicola, E. cichoracearum, and Podosphaera hibiscicola, with a sharp increase during the conidia senescence in E. quercicola. Additionally, the main spectral excitation detected by fluorescence spectrometery was 375 nm for these species, with a common emission peak at approximately 458-463 nm, and an additional trend at 487 nm for P. hibiscicola. Because NAD(P)H has a similar spectral feature, we further investigated the relation between NAD(P)H and conidial autofluorescence by fluorescence spectra. We observed that the reduced coenzymes prominently contributed to conidial autofluorescence; however, the conidial autofluorescence in P. hibiscicola displayed a different trend that may be affected by the oxidized coenzyme -NAD. Finally, the normalized average spectra of these three powdery mildew species and standard samples showed that the spectral trend of each species was similar but that the features in detail were specific and distinct based on principal component analysis. In conclusion, we showed and characterized conidial autofluorescence in three powdery mildew species for the first time. The specific conidial autofluorescence in these species provides a new idea for the development of field spore capture and identification devices for the discrimination of powdery mildew at the species level.

The Transcription Factor CsAtf1 Negatively Regulates the Cytochrome P450 Gene CsCyp51G1 to Increase Fludioxonil Sensitivity in Colletotrichum siamense.

Previous studies have shown that the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK) signaling pathway and its downstream transcription factor CsAtf1 are involved in the regulation of fludioxonil sensitivity in C. siamense. However, the downstream target genes of CsAtf1 related to the fludioxonil stress response remain unclear. Here, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and high-throughput RNA-sequencing (RNA-Seq) to identify genome-wide potential CsAtf1 target genes. A total of 3809 significantly differentially expressed genes were predicted to be directly regulated by CsAtf1, including 24 cytochrome oxidase-related genes. Among them, a cytochrome P450-encoding gene, designated CsCyp51G1, was confirmed to be a target gene, and its transcriptional expression was negatively regulated by CsAtf1, as determined using an electrophoretic mobility shift assay (EMSA), a yeast one-hybrid (Y1H) assay, and quantitative real-time PCR (qRT-PCR). Moreover, the overexpression mutant CsCYP51G1 of C. siamense exhibited increased fludioxonil tolerance, and the CsCYP51G1 deletion mutant exhibited decreased fludioxonil resistance, which revealed that CsCyp51G1 is involved in fludioxonil sensitivity regulation in C. siamense. However, the cellular ergosterol content of the mutants was not consistent with the phenotype of fludioxonil sensitivity, which indicated that CsCyp51G1 regulates fludioxonil sensitivity by affecting factors other than the ergosterol level in C. siamense. In conclusion, our data indicate that the transcription factor CsAtf1 negatively regulates the cytochrome P450 gene CsCyp51G1 to increase fludioxonil sensitivity in C. siamense.

Ectopic Expression of HbRPW8-a from Hevea brasiliensis Improves Arabidopsis thaliana Resistance to Powdery Mildew Fungi (Erysiphe cichoracearum UCSC1).

The RPW8s (Resistance to Powdery Mildew 8) are atypical broad-spectrum resistance genes that provide resistance to the powdery mildew fungi. Powdery mildew of rubber tree is one of the serious fungal diseases that affect tree growth and latex production. However, the RPW8 homologs in rubber tree and their role of resistance to powdery mildew remain unclear. In this study, four RPW8 genes, HbRPW8-a, b, c, d, were identified in rubber tree, and phylogenetic analysis showed that HbRPW8-a was clustered with AtRPW8.1 and AtRPW8.2 of Arabidopsis. The HbRPW8-a protein was localized on the plasma membrane and its expression in rubber tree was significantly induced upon powdery mildew infection. Transient expression of HbRPW8-a in tobacco leaves induced plant immune responses, including the accumulation of reactive oxygen species and the deposition of callose in plant cells, which was similar to that induced by AtRPW8.2. Consistently, overexpression of HbRPW8-a in Arabidopsis thaliana enhanced plant resistance to Erysiphe cichoracearum UCSC1 and Pseudomonas syringae pv. tomato DC30000 (PstDC3000). Moreover, such HbRPW8-a mediated resistance to powdery mildew was in a salicylic acid (SA) dependent manner. Taken together, we demonstrated a new RPW8 member in rubber tree, HbRPW8-a, which could potentially contribute the resistance to powdery mildew.

First Report of leaf spot on Cucumber Caused by Pantoea ananatis in Hainan of China.

Cucumber (Cucumis sativus L.) is one of the most important vegetables cultivated in the world. It is widely cultivated and mostly grown under greenhouse conditions (Sallam et al. 2021). Cucumber has a long growth cycle and is particularly susceptible to bacterial diseases. In May 2021, bacterial leaf spot was found on cucumbers of the variety Lyuyou NO.3 in Hainan Province, China. In the early stage of the disease, the leaves showed small yellow-brown spots in the shape of water stains. When exposed to light, a yellow halo around the disease spots could be seen. In later stages, the lesions gradually become larger and more yellow. The leaf veins around the disease site also gradually turned yellow (Figure 2a). In serious cases, the whole leaf turned yellow, resulting in leaf death. We collected plants with the same symptoms from 25 different farms in Hainan Province. Five plants were selected from each farm by the classic five-point sampling method and three leaves were selected from each plant, for a total of 15 leaves collected from each farm. Then three leaves were randomly selected from the 15 leaves on each farm for isolation of the pathogen, and a total of 75 leaves were isolated. We found that the incidence of the disease was from 20% to 30% based on a diagnostic test, which conducted on 75 cucumber leaves samples suspected of same symptom of cucumber, collected from Hainan Province. Using microscopy, bacterial streaming was observed to tentatively identify the causal agent as a bacteria. Tissue isolation was used to isolate the responsible pathogens. A 5 mm × 5 mm sample of tissue at the junction of diseased and healthy sections was collected. First, the surface of the tissue was disinfected in a 75% ethanol solution for 30 sec; then it was soaked in 2% NaOCl for 5-7 min, and finally, it was washed thrice in sterile distilled water. The tissues were inoculated onto lysogen broth culture media (LB) and cultured in a 28℃ incubator for 2 days. Bacterial colonies that emerged from the tissues were cultured in LB. Four isolated colonies were selected for verification. The colonies of isolated from the diseased leaves of cucumber are round, egg yellow and slightly sticky (Figure 2c). The isolate named PA-1 was identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F/1492R (Lane 1991) and gyrB gene (Li et al. 2019). Sequences were stored in GenBank with the accession numbers OK576932.1 (16S rRNA, PA-1) and OL978577 (gyrB); BLASTn was used to compare these with other GenBank sequences. Sequencing of the 16S rRNA gene showed that PA-1 had a sequence length of 1403bp, with 99.78% genetic similarity to Pantoea ananatis strain MZ007857.1. Sequencing of the gyrB gene showed that the sequence length of PA-1 was 1136bp, with 99.29% genetic similarity to P. ananatis strain MW981331.1. Then, a pathogenicity text was conducted to verify Koch's postulates, which was done by first inoculating P. ananatis into LB liquid medium (shake culture at 28°C, 180 r/min). The log phase cell was collected by centrifugation (5,000 r/min for 2 min at 4°C), and inoculated strains were resuspended in sterile water at OD600 = 0.5. The bacterial suspension was inoculated on healthy cucumber leaves with a syringe. The control was sterile water, which was injected onto healthy cucumber leaves using the same methodology. The plants were placed in a greenhouse with a diurnal temperature difference of 21- 27°C and were observed daily. After two weeks, all bacterial inoculated plants developed symptoms of shriveling and necrosis (Figure 2b), while the control group showed no symptoms. From the symptomatic plants, the pathogen was isolated again and identified by morphological and molecular characterization. The sequences of the isolates recovered from the inoculated experiment matched 100% the sequences of the isolate PA-1. Koch's postulates were completed by following the previously described method. To our knowledge, this is the first report of P. ananatis causing leaf spot of cucumber.

The Colletotrichum siamense Hydrophobin CsHydr1 Interacts with the Lipid Droplet-Coating Protein CsCap20 and Regulates Lipid Metabolism and Virulence.

Previous studies of the lipid droplet-coating protein Cap20 in Colletotrichum show that it plays a key role in appressorium development and virulence. In this study, the hydrophobin CsHydr1, which contains a signal peptide of 19 amino acids and a hydrophobic domain (HYDRO), was shown to interact with CsCap20 in Colletotrichum siamense. The CsHydr1 deletion mutant showed slightly enhanced mycelial growth, small conidia, slow spore germination and appressoria formation, cell wall integrity and virulence. Like CsCAP20, CsHydr1 is also localized on the lipid droplet surface of C. siamense. However, when CsCap20 was absent, some CsHydr1 was observed in other parts. Quantitative lipid determination showed that the absence of either CsHydr1 or CsCap20 reduced the content of lipids in mycelia and conidia, while the effect of CsCap20 was more obvious; these results suggest that an interaction protein CsHydr1 of CsCap20 is localized on the lipid droplet surface and involved in lipid metabolism, which affects appressorium formation and virulence in C. siamense.

First Report of Stemphylium lycopersici Causing Leaf Spot on Hevea brasiliensis in China.

Hevea brasiliensis is widely planted in tropical and subtropical regions and is the main source of natural rubber production. The growth of rubber trees is plagued by various leaf diseases, resulting in decreased rubber production. From January to March in 2020, a severe leaf spots disease on Hevea brasiliensis found in Agricultural Science Base in Haidian campus of Hainan University (20° 03' 31″ N, 110° 19' 07″ E), Haikou, Hainan province, China. Spots were only observed on the mature green rather than young and bronze-colored leaves. This symptom has never been reported on the leaves of Hevea brasiliensis. During the early stages of the disease, gray leaf spots were concentrated to the leaf margins, but later expanded forming irregular gray lesions with chlorotic edges (Figure 1A). Eventually, lesions became necrotic shot holed, and leaves curled, wilted, and dropped. Five small pieces were cut from the margin of spots from different infected leaves, and were surface disinfected with 75% alcohol three times for five seconds each time and 1% sodium hypochlorite solution (NaClO) for 60 s. After washing twice with sterile water, leaf pieces were placed in the center of plates with Potato Dextrose Agar (PDA) medium and incubated for one week at 28 °C. After 7 days, mycelium developed and colonies were single-spore cultured for further study. One of the strains labeled HN01 developed a yellowish-brown to reddish-brown pigment on PDA, and the colonies were gray and cottony. The colony and pigment feature very consistent with Stemphylium sp. (Figure 2) (Li et al. 2017). Conidiophore were solitary, transparent to pale, mostly 102.1-228.8 µm × 4.0-5.8 µm, with 2-3 septa and apical vesicular swellings 6.5-7.9 µm. The dimensions of conidia were 28.3-45.1 × 11.5-17.5 µm and one septum (Figure 3). Conidia of S. lycopersici were solitary, oblong with a conical end at the apex, with 1-2 septa, and constricted at the transverse septum. The internal transcribed spacer region of rDNA was amplified with primers ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3'/5'-TCCTCCGCTTATTGATATGC-3'), glyceraldehyde-3-phosphate dehydrogenase (gpd) was amplified with primers GPD-F/R (5'-GCACCGACCACAAAAATC-3'/ 5'-GGGCCGTCAACGACCTTC-3'), calmodulin region (cmdA) was amplified with the primers CALDF1/CALDR2 (5'-AGCAAGTCTCCGAGTTCAAGG-3'/5'-CTTCTGCATCATCAYCTGGACG3') from genomic DNA of strain HN01 (Xie et al. 2018), and PCR products were sequenced. The ITS sequence of strain HN01 (GenBank Accession No. MZ496930) had 99.64% identity with isolates sl001, sl110, sl111, and sl112 of Stemphylium lycopersici (GenBank Accession No. KX858848.1, MF480547.1, MF480548.1, MF480549.1). Similarly GPD sequences (GenBank Accession No. MZ505106) had 100% identity with strain xiqing, HZ2114 and HZ2115 of Stemphylium lycopersici (GenBank Accession No. KR911809.1, KR911810.1, KT957742.1 and KT957743.1), and CMDA sequences (GenBank Accession No. MZ505105) had 99.85% identity with Stemphylium lycopersici strain LJ1609270201 (GenBank Accession No. MG742412.1). A phylogenetic analysis constructed by MEGA6.0 based on concatenated sequences of the HN01 and another 17 strains from GenBank by using the maximum-likelihood (ML) method showed that the HN01 was clustered and matched with Stemphylium lycopersici LJ1609270201 (Figure 4). To satisfy Koch's postulates, we inoculated mature green leaves of Hevea brasiliensis with mycelial plugs (diameter = 5 mm) of pure cultured strain HN01. All leaves of Hevea brasiliensis were wrapped in a freezer bag to maintain relative humidity >85%, and the temperature of greenhouse is 28ºC. The disease developed on the inoculated leaves after 2-3 days, but not on control leaves (Figure 1B). We used the same method as before to re-isolate the pathogen, which had the same morphology and genotypes as the original isolate. S. lycopersici has been reported to infect the leaves of a variety of plants, including pepper, tomato, eggplant, watermelon, Physalis alkekengi. (Yang et al.2017; Ben et al. 2017; Yang et al. 2020). To our knowledge, this is the first record of S. lycopersici causing leaf spot of Hevea brasiliensis in China, and Hevea brasiliensis is the global new host of S. lycopersici. Hevea brasiliensis is the main source of natural rubber and is widely planted in southern China. Therefore, it is imperative to implement disease management measures to prevent potential threats.

First report of crown gall disease caused by Agrobacterium radiobacter on olive plants in Layyah, Punjab, Pakistan.

Crown galls were observed on one-year-old olive plants (Olea europaea cv. Manzanilla) in the District Layyah (30.9693° N, 70.9428° E) of Punjab, Pakistan. Large tumors were evident on collars region, causing growth stunting, leaf yellowing, and overall plant dieback (Supplementary fig. 1). Total 900 of olive plant were grown including 300 young plants in five hectare orchards, around 25% of the young plants in orchard had gall formation with varying in size (2-15cm), majority of the infected plants were grown near the water channel, where soil moisture level were high (90-100%). Other olive orchards in the same area have not crown gall problem and the tumorigenic strains of bacteria can cause crown gall on plants (Nemanja Kuzmanovic et al. 2015). This study was aimed to determine the pathogen of disease. The randomized collected samples were rinsed with tap water and galls were sterilized with 10% sodium hypochlorite solution for 1.5-3.0 min, washed with sterilized Distilled Water (SDW) then chopped and immersed overnight in SDW at room temperature. Isolations were carried out by plating the internal gall tissues on fresh Luria Bertani agar (LB agar) supplemented with natamycin. After incubating at 28°C for 5 days, 10 single colonies were transferred on new LBA plates for further cultivation at 28°C. After 48 to 72 h, three strains showed white to cream-colored, smooth, convex, glistening, circular with entire edges, and mucoid bacterial colonies resembling Agrobacterium spp. These three strains (BAT01, BAT02, BAT03) also showed biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. They were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with CaCO3 (Young et al. 2015). Amplification and sequencing of these three strain's 16S rRNA region and chromosomal recA gene with the universal primers fD1/rP2 and F2898/F2899 verified the identification at species level (Weisburg et al. 1991) . BLAST analysis revealed 100% identity for 16S rRNA and recA gene between the olive crown gall strains. Accession No. of deposited sequences were given in table 1 and the reference sequences GenBank Accessions No. of A. tumefaciens is FM209485.1 and KY913787 respectively. Phylogenetic analysis based on 16S rRNA of the strains from the crown gall and reference strains of various species of Agrobacterium by Maximum-likelihood method with Tamura's three-parameter model using the MEGA X software program confirmed the strain from olive was A. tumefaciens (Supplementary fig. 2). Inoculating the crown part of the plant through wounds of sterile needles plunged into young (2 to 3 day) bacterial culture (107 CFU/ml) and sterile distilled water (SDW) was screened for pathogens on 10 one-year-old olive plants cv. Manzanilla. Plants were grown at 23 ± 3°C, and tumor formation was observed 4 weeks after inoculation. Typical tumours formed and no symptoms found in control plants at inoculation sites and Koch's postulates were fulfilled with re-isolation and amplification of bacteria with recA gene region. This data shows that A. tumefaciens causes crown gall in olive plants. though it is reported before in different olive growing region in the world but This is first time reported in Layyah, Punjab, Pakistan.