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.

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.

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 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.

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.

CsAtf1, a bZIP transcription factor, is involved in fludioxonil sensitivity and virulence in the rubber tree anthracnose fungus Colletotrichum siamense.

In phytopathogenic fungi, the HOG MAPK pathway has roles in osmoregulation, fungicide sensitivity, and other processes. The ATF1/CREB-activating transcription factor Atf1 is a regulator that functions downstream of the HOG MAPK pathway. Here, we identified a gene, designated CsAtf1, that encodes a bZIP transcription factor in Colletotrichum siamense, which is the main pathogen that causes Colletotrichum leaf fall disease in rubber trees in China. CsAtf1 localizes to the nucleus. Its mRNA expression correlates positively with that of CsPbs2 and CsHog1 in the HOG MAPK pathway in response to activator (anisomycin), inhibitor (SB203580) and fludioxonil treatments. The CsAtf1 deletion mutant showed slightly retarded mycelial growth, small conidia, slow spore germination, and abnormal appressorium formation. This mutant showed the increased spore germination rate after fludioxonil treatment and more resistance to the fungicide fludioxonil than did the wild-type fungus. However, unlike deletion of Pbs2 or Hog1, which resulted in greater sensitivity to osmotic stress, the CsAtf1 deletion induced slightly increased resistance to osmotic stress and the cell wall stress response. The ΔCsAtf1 strain also exhibited significantly reduced virulence on rubber tree leaves. These data revealed that CsAtf1 plays a key role in the regulation of fludioxonil sensitivity and in pathogenicity regulation in C. siamense.

A putative effector of the rubber-tree powdery mildew fungus has elicitor activity that can trigger plant immunity.

MAIN CONCLUSION: A putative powdery mildew effector can elicit defense responses including reactive oxygen species and callose accumulations in model plants Nicotiana benthamiana and Arabidopsis thaliana and host plant Hevea brasiliensis. Powdery mildew fungi cause severe diseases in many agricultural plants, such as the mildew fungus Erysiphe quercicola infecting the rubber tree (Hevea brasiliensis), causing latex yield losses. However, effectors of E. quercicola were rarely functionally characterized. In this study, we identified a highly specific candidate-secreted effector protein, EqCSEP04187, from E. quercicola. This putative effector is expressed at the late stage but not the early stage during infection. The constitutive expression of EqCSEP04187 in model plants Nicotiana benthamiana and Arabidopsis thaliana elicited defense responses, as did transient expression of EqCSEP04187 in protoplasts of H. brasiliensis. Introducing EqCSEP04187 into another H. brasiliensis-associated fungal pathogen, Colletotrichum gloeosporioides, inhibited H. brasiliensis infection, and infection by E. quercicola was decreased in the A. thaliana eds1 mutant expressing EqCSEP04187. Further analysis suggests that these reductions in infection were the consequences of EqCSEP04187 eliciting defense responses. Our study suggests that this putative effector has elicitor activity that can improve plant resistance.

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.

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.

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.

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

HbLFG1, a Rubber Tree (Hevea brasiliensis) Lifeguard Protein, Can Facilitate Powdery Mildew Infection by Suppressing Plant Immunity.

Powdery mildew causes substantial losses in crop and economic plant yields worldwide. Although powdery mildew infection of rubber trees (Hevea brasiliensis), caused by the biotrophic fungus Erysiphe quercicola, severely threatens natural rubber production, little is known about the mechanism by which E. quercicola adapts to H. brasiliensis to invade the host plant. In barley and Arabidopsis thaliana, lifeguard (LFG) proteins, which have topological similarity to BAX INHIBITOR-1, are involved in host plant susceptibility to powdery mildew infection. In this study, we characterized an H. brasiliensis LFG protein (HbLFG1) with a focus on its function in regulating defense against powdery mildew. HbLFG1 gene expression was found to be upregulated during E. quercicola infection. HbLFG1 showed conserved functions in cell death inhibition and membrane localization. Expression of HbLFG1 in Nicotiana benthamiana leaves and A. thaliana Col-0 was demonstrated to significantly suppress callose deposition induced by conserved pathogen-associated molecular patterns chitin and flg22. Furthermore, we found that overexpression of HbLFG1 in H. brasiliensis mesophyll protoplasts significantly suppressed the chitin-induced burst of reactive oxygen species. Although A. thaliana Col-0 and E. quercicola displayed an incompatible interaction, Col-0 transformants overexpressing HbLFG1 were shown to be susceptible to E. quercicola. Collectively, the findings of this study provide evidence that HbLFG1 acts as a negative regulator of plant immunity that facilitates E. quercicola infection in H. brasiliensis.

The stability of the coiled-coil structure near to N-terminus influence the heat resistance of harpin proteins from Xanthomonas.

BACKGROUND: Heat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance. RESULTS: Each of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1. CONCLUSIONS: The stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.

HpaXpm, a novel harpin of Xanthomonas phaseoli pv. manihotis, acts as an elicitor with high thermal stability, reduces disease, and promotes plant growth.

BACKGROUND: Harpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen-plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown. RESULTS: We identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two ß-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures. CONCLUSIONS: These findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature.

Use of an Infectious cDNA Clone of Pepper Veinal Mottle Virus to Confirm the Etiology of a Disease in Capsicum chinense.

The pepper cultivar Yellow Lantern, one of the spiciest pepper varieties, is a local germplasm of Capsicum chinense, cultivated exclusively on Hainan Island, China. However, this variety is susceptible to viral diseases that severely affect its production. In this study, we report that pepper veinal mottle virus (PVMV) is associated with foliar chlorosis and rugosity symptoms in Yellow Lantern. To verify this correlation, we constructed a full-length cDNA clone of a PVMV isolate named HNu. The virus progeny derived from the cDNA clone replicated and moved systemically in the pepper, inducing the same symptoms as those induced by PVMV-HNu in Yellow Lantern peppers in the field. The results support that PVMV-HNu is the causal agent of foliar chlorosis and rugosity disease in Yellow Lantern. This knowledge will help in the diagnosis and prevention of disease caused by PVMV. Furthermore, the cDNA clone serves as a reverse genetic tool to study the molecular pathogenesis of PVMV.

Antibacterial activity and rice-induced resistance, mediated by C15surfactin A, in controlling rice disease caused by Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is an important pathogen in rice. C15surfactin A, produced by Bacillus velezensis HN-2, displayed antibacterial activity against Xoo and effectively inhibited its infection of rice. The median inhibitory concentration of C15surfactin A was 9.27 µg/mL. Scanning electron and transmission electron microscopy examination showed that C15surfactin A caused significant damage to the cell wall structure of Xoo cells. On the other hand, dramatic increases in the activity of phenylalanine ammonia-lyase (Pal) and H2O2 content were observed in rice leaves inoculated with Xoo from 0 h to 72 h. Quantitative PCR assays indicated that C15surfactin A exposure upregulated the expression of the genes Pr1a, CatA, and Pal. The results showed that C15surfactin A could inhibit the growth of Xoo and effectively induce rice resistance to Xoo by triggering a hypersensitive reaction (HR) via mediation of the activities of antioxidant-related enzymes. Taken together, C15surfactin A has strong antibiotic activity against Xoo and effectively induces rice resistance to Xoo. These results highlight the potential of C15surfactin A as a biocontrol agent against Xoo in rice.

Antifungal mechanism of bacillomycin D from Bacillus velezensis HN-2 against Colletotrichum gloeosporioides Penz.

Anthracnose is a leaf spot, blossom blight, or fruit rot disease caused by Colletotrichum gloeosporioides (Penz.). It is the most prevalent disease in mango-growing countries worldwide. Lipopeptides, such as those in the iturin family, account for the majority of antifungal secondary metabolites in Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus velezensis, and includes bacillomycin D. Thus far, the mechanism of bacillomycin D's activity has not been clear. In this study, bacillomycin D was isolated from B. velezensis HN-2, which strongly inhibits C. gloeosporioides (Penz.). The median inhibitory concentration of bacillomycin D was 2.162 µg/mL, causing deformation and damage to C. gloeosporioides (Penz.). Bacillomycin D showed more potent activity against C. gloeosporioides (Penz.) than two common fungicides prochloraz and mancozeb. Scanning and transmission electron microscopy revealed that bacillomycin D could injure the cell wall and cell membrane of the hyphae and spores of C. gloeosporioides (Penz.), and the cytoplasm and organelles inside the cell were exuded and formed empty holes. This research clarifies the mechanism underlying bacillomycin D antifungal activity and reveals its high potential as a biopesticide to control phytopathogens.

Complete genomic sequence and organization of a novel mycovirus from Phoma matteuccicola strain LG915.

The complete genome of a double-stranded RNA (dsRNA) mycovirus, Phoma matteuccicola partitivirus 1 (PmPV1) was sequenced. It consists of two dsRNA segments, 1664 bp (dsRNA-1) and 1383 bp (dsRNA-2) in length, each containing a single open reading frame (ORF) potentially encoding a 46.78-kDa protein and a 40.92-kDa protein, respectively. dsRNA-1 encodes a putative polypeptide with a conserved RNA-dependent RNA polymerase (RdRp) domain that shows sequence similarity to the corresponding proteins of partitiviruses. The protein encoded by dsRNA-2 has no significant similarity to the typical coat proteins (CPs) of partitiviruses, but structure analysis nevertheless suggested that it might function as a coat protein. Purified viral particles of PmPV1 were isometric and approximately 29 nm in diameter. Phylogenetic analysis showed that PmPV1 is closely related to members of the genus Gammapartitivirus within the family Partitiviridae but forms a separate branch with Colletotrichum acutatum RNA virus 1 and Ustilaginoidea virens partitivirus 2. This is the first report of the full-length nucleotide sequence of a novel virus of the genus Gammapartitivirus infecting P. matteuccicola strain LG915, the causal agent of leaf blight of Curcuma wenyujin.

Areca Palm Necrotic Ringspot Virus, Classified Within a Recently Proposed Genus Arepavirus of the Family Potyviridae, Is Associated With Necrotic Ringspot Disease in Areca Palm.

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.