A protein elicitor PeVn1 from Verticillium nonalfalfae HW recognized as a MAMP triggers plant immunity response.

Protein elicitors can induce plant systemic resistance to pathogens. The recognition of a potential elicitor activates intracellular signaling events, leading to plant resistance against pathogens. In this study, a novel protein elicitor was isolated from the culture filtrate of Verticillium nonalfalfae and named PeVn1, which can induce cell death in several plant species. The PeVn1 gene was then cloned and expressed in Escherichia coli. The recombinant protein PeVn1 triggers cell death in Nicotiana benthamiana in NbBAK1 and NbSOBIR1 dependent manner. Through bioassay analysis showed that the recombinant PeVn1 induced early defense induction events, such as reactive oxygen species burst, callose deposition and the activation of defense hormone signaling pathways and defense enzyme activities. Moreover, PeVn1 significantly enhanced resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum, Botrytis cinerea and N. benthamiana mosaic virus and tomato to Pseudomonas syringae pv. Tomato DC3000. In conclusion, our study reveals that PeVn1 protein as a microbe-associated molecular pattern can induce plant immune responses, which provides a theoretical basis for the development of novel protein-induced disease resistance agents.

Potential Candidate Molecule of Photosystem II Inhibitor Herbicide-Brassicanate A Sulfoxide.

Brassicanate A sulfoxide, a secondary metabolite of broccoli, exhibited the inhibition of weed growth, but its mechanism of action on weeds remains unclear. To elucidate the mechanism by which brassicanate A sulfoxide suppresses weeds, this study explores the interaction between brassicanate A sulfoxide and the photosystem II D1 protein through molecular docking and molecular dynamics simulations. This research demonstrates that brassicanate A sulfoxide interacts with the photosystem II D1 protein by forming hydrogen bonds with Phe-261 and His-214. The successful expression of the photosystem II D1 protein in an insect cell/baculovirus system validated the molecular docking and dynamics simulations. Biolayer interferometry experiments elucidated that the affinity constant of brassicanate A sulfoxide with photosystem II was 2.69 × 10-3 M, suggesting that brassicanate A sulfoxide can stably bind to the photosystem II D1 protein. The findings of this study contribute to the understanding of the mode of action of brassicanate A sulfoxide and also aid in the development of natural-product-based photosynthesis-inhibiting herbicides.

Predicting potential distribution of the Rhinoncus sibiricus under climatic in China using MaxEnt.

In recent years, buckwheat (Fagopyrum spp.) is being increasingly damaged by the Siberian tortoise beetle (Rhinoncus sibiricus Faust). Adults and nymphs feed on leaf tissues and caulicles, thus damaging its stems and leaves. In this study, we investigated the habits, distribution, and environmental impact of R. sibiricus using MaxEnt, an ecological niche model. Geographic information about the infestation site from previous field surveys and climatic data from 2013 to 2018 were organized and optimized using R. The impact factors were calculated using MaxEnt software. The results indicate that population fluctuations in R. sibiricus are related to changes in temperature, humidity, and their spatial distribution. Under current climatic conditions, R. sibiricus is mainly distributed in northern China, with sporadic distribution in south-western China. The values for a survival probability threshold > 0.3 were: precipitation during the wettest month (bio13), 70.31-137.56 mm; mean temperature of the coldest quarter (bio11), -15.00-0.85°C; mean temperature of the warmest quarter (bio10), 11.88-23.16°C; precipitation during the coldest quarter (biol9), 0-24.39 mm. The main factors contributing > 70% to the models were precipitation during the wettest month and coldest quarter, and mean temperature during the warmest and coldest quarters. Under both future climate models, the center of the fitness zone moves northward. Our results will be useful in guiding administrative decisions and support farmers interested in establishing control and management strategies for R. sibiricus. This study could also serve as a reference for future research on other invasive pests.

The Aqueous Extract of Brassica oleracea L. Exerts Phytotoxicity by Modulating H2O2 and O2- Levels, Antioxidant Enzyme Activity and Phytohormone Levels.

Allelopathic interactions between plants serve as powerful tools for weed control. Despite the increasing understanding of the allelopathic mechanisms between different plant species, the inhibitory effects of B. oleracea on weed growth remain poorly understood. In this study, we conducted experiments to demonstrate that B. oleracea extract can suppress the germination of Panicum miliaceum L.varruderale Kit. seeds as well as of the roots, shoots and hypocotyl elongation of P. miliaceum seedlings. Furthermore, we observed that B. oleracea extract reduced the levels of hydrogen peroxide and superoxide anion in the roots while increasing the activities of catalase and ascorbate peroxidase. In the shoots, B. oleracea extract enhanced the activities of superoxide dismutase and peroxidase. Moreover, the use of the extract led to an increase in the content of phytohormones (indole-3-acetic acid, indole-3-acetaldehyde, methyl indole-3-acetate, N6-isoPentenyladenosine, dihydrozeatin-7-glucoside, abscisic acid and abscisic acid glucose ester) in P. miliaceum seedlings. Interestingly, the aqueous extract contained auxins and their analogs, which inhibited the germination and growth of P. miliaceum. This may contribute to the mechanism of the B. oleracea-extract-induced suppression of P. miliaceum growth.

Isolation and Identification of Herbicidal Active Compounds from Brassica oleracea L. and Exploration of the Binding Sites of Brassicanate A Sulfoxide.

Brassica oleracea L. has strong allelopathic effects on weeds. However, the allelochemicals with herbicidal activity in B. oleracea L. are still unknown. In this study, we evaluated the activity of allelochemicals isolated from Brassica oleracea L. based on the germination and growth of model plant Lactuca sativa Linn., grass weed Panicum miliaceum, and broadleaf weed Chenopodium album. Additionally, we employed molecular docking to predict the binding of brassicanate A sulfoxide to herbicide targets. The results of this study showed that eight compounds with herbicidal activity were isolated from B. oleracea L., and the predicted results indicated that brassicanate A sulfoxide was stably bound to dihydroxyacid dehydratase, hydroxymethylpyruvate dioxygenase, acetolactate synthase, PYL family proteins and transport inhibitor response 1. This research provides compound sources and a theoretical foundation for the development of natural herbicides.

Characterization of a Bacillus subtilis S-16 Thiazole-Synthesis-Related Gene thiS Knockout and Antimicrobial Activity Analysis.

Bacillus subtilis S-16 isolated from sunflower-rhizosphere soil is an effective biocontrol agent for preventing soilborne diseases in plants. Previous research revealed that the volatile organic compounds (VOCs) produced by the S-16 strain have strong inhibitory effects on Sclerotinia sclerotiorum. The identification of the VOCs of S-16 using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed 35 compounds. Technical-grade formulations of four of these compounds were chosen for further study: 2-pentadecanone, 6,10,14-trimethyl-2-octanone, 2-methyl benzothiazole (2-MBTH), and heptadecane. The major constituent, 2-MBTH, plays an important role in the antifungal activity of the VOCs of S-16 against the growth of Sclerotinia sclerotiorum. The purpose of this study was to determine the impact of the thiS gene's deletion on the 2-MBTH production and to conduct an antimicrobial activity analysis of the Bacillus subtilis S-16. The thiazole-biosynthesis gene was deleted via homologous recombination, after which the contents of 2-MBTH in the wild-type and mutant S-16 strains were analyzed using GC-MS. The antifungal effects of the VOCs were determined using a dual-culture technique. The morphological characteristics of the Sclerotinia sclerotiorum mycelia were examined via scanning-electron microscopy (SEM). Additionally, the lesion areas on the sunflower leaves with and without treatment with the VOCs from the wild-type and mutant strains were measured to explore the effects of the VOCs on the virulence of the Sclerotinia sclerotiorum. Moreover, the effects of the VOCs on the sclerotial production were assessed. We showed that the mutant strain produced less 2-MBTH. The ability of the VOCs produced by the mutant strain to inhibit the growth of the mycelia was also reduced. The SEM observation showed that the VOCs released by the mutant strain also caused more flaccid and gapped hyphae in the Sclerotinia sclerotiorum. The Sclerotinia sclerotiorum treated by the VOCs produced by the mutant strains caused more damage to the leaves than that treated by the VOCs produced by the wild type and the mutant-strain-produced VOCs inhibited sclerotia formation less. The production of 2-MBTH and its antimicrobial activities were adversely affected to varying degrees by the deletion of thiS.

RpoZ regulates 2,4-DAPG production and quorum sensing system in Pseudomonas fluorescens 2P24.

Introduction: Pseudomonas fluorescens 2P24 was isolated from soil of natural decay associated with wheat take-all and it can effectively control soil-borne diseases caused by a variety of plant pathogens. 2,4-diacetylphloroglucinol (2,4-DAPG), is produced by P. fluorescens 2P24 and plays an important role in the prevention and control of plant diseases. To understand the resistant mechanism, in this study, we conducted experiments to explore the regulation role of rpoZ in the synthesis of the antibiotic 2,4-DAPG and regulation of QS system. Methods: A random mini-Tn5 mutagenesis procedure was used to screen regulators for phlA transcription in stain PM901, which containing a phlA∷lacZ transcriptional fusion reporter plasmid. We identified 12 insertion mutants could significantly change phlA gene expression. By analyzing the amino acid sequences of the interrupted gene, we obtained a mutant strain Aa4-29 destroyed the rpoZ gene, which encodes the omiga subunit. We constructed the plasmid of rpoZ mutant (pBBR-△rpoZ) transformed into competent cells of P. fluorescens 2P24 by electro-transformation assay. The strains of P. fluorescens 2P24/pBBR, 2P24-△rpoZ/pBBR, 2P24-△rpoZ/pBBR-rpoZ were used to evaluate the regulation role of rpoZ in 2,4-DAPG production and quorum sensing system. Results: According to ß-galactosidase activity, we found that rpoZ positively regulated the expression of phlA (a synthesis gene of 2,4-DAPG) and PcoI (a synthesis gene of PcoI/PcoR QS signal system) at the transcriptional level. The production of 2,4-DAPG antibiotic and signal molecule AHL was influenced by rpoZ. Further, rpoZ was involved in regulating rsmA expression. RpoZ also has a certain regulatory effect on rpoS transcription, but no effect on the transcription of phlF, emhABC and emhR. According to the biocontrol assay, P. fluorescens 2P24 strains with rpoZ showed obvious antagonism ability against the Rhizoctonia solani in cotton, while the mutant strain of rpoZ lost the biocontrol effect. RpoZ had a significant effect on the swimming and biofilm formation in P. fluorescens 2P24. Conclusion: Our data showed that rpoZ was an important regulator of QS system, 2,4-DAPG in P. fluorescens 2P24. This may imply that P. fluorescens 2P24 has evolved different regulatory features to adapt to different environmental threats.

Genomic and metabolic features of Bacillus cereus, inhibiting the growth of Sclerotinia sclerotiorum by synthesizing secondary metabolites.

We investigated the biocontrol mechanism of Bacillus cereus CF4-51 to find powerful microbes that effectively control Sclerotinia sclerotiorum. To assess its inhibitory effect on fungal growth, the plant pathogen (S. sclerotiorum) was co-cultured with Bacillus cereus. Scanning electron microscope (SEM) was used to study the morphology of S. sclerotiorum treated with CF4-51 biofumigant. The expression of sclerotium formation-related genes was analyzed by qRT-PCR. We performed whole genome sequencing of CF4-51 by PacBio Sequel platform. Lipopeptides were extracted from strain CF4-51 according to the method of hydrochloric acid precipitation and methanol dissolution. The volatiles CF4-51 were identified using gas chromatography-mass spectrometry (GC-MS). We found that the volatile organic compounds (VOCs) released by CF4-51 damaged the S. sclerotiorum hyphae and inhibited the formation of sclerotia. The qRT-PCR data revealed the down-regulated expression of the genes involved in sclerotial formation. Moreover, we analyzed the B. cereus CF4-51 genome and metabolites. The genome consisted of 5.35 Mb, with a GC content of 35.74%. An examination of the genome revealed the presence of several gene clusters for the biosynthesis of antibiotics, siderophores, and various other bioactive compounds, including those belonging to the NRPS-like, LAP, RIPP-like, NRPS, betalactone, CDPS, terpene, ladderane, ranthipeptide, and lanthipeptide (class II) categories. A gas chromatography-tandem mass spectrometry analysis identified 45 VOCs produced by strain CF4-51. Among these, technical grade formulations of five were chosen for further study: 2-Pentadecanone, 6,10,14-trimethyl-,1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester, Dibutyl phthalate, Cyclododecane, Heptadecane. the five major constituents play important roles in the antifungal activity of the VOCs CF4-51 on the growth of S. sclerotiorum. The secondary metabolites produced by strain CF4-51are critical for the inhibition of S. sclerotiorum hyphal growth and sclerotial formation.

Whole-genome analysis revealed the growth-promoting mechanism of endophytic bacterial strain Q2H1 in potato plants.

Introduction: Endophytes are non-pathogenic inhabitants of healthy plant tissues and have been found to promote plant growth and health. The endophytic bacterial strain Q2H1 was isolated from the roots of the potato and was identified to exhibit growth-promoting effects in potato plants. Methods: Whole-genome sequencing was performed to reveal the mechanism underlying its growth-promoting effect. The obtained sequencing data of approximately 5.65 MB encompassed 5,533 coding sequences. Of note, nine secondary metabolite gene clusters, including siderophore gene clusters, closely associated with plant growth promotion (PGP) were predicted by antiSMASH software. Comparative genomic analysis revealed that Q2H1 belongs to the genus Peribacillus. By gene function annotation, those genes related to plant growth-promoting activities, including indole-3-acetic acid (IAA) synthesis in tryptophan metabolism, siderophore biosynthetic activity, phosphate solubilization, nitrogen fixation, and related genes, were summarized. IAA (14.4 µg/ml) was presumptively produced by Q2H1 using the Salkowski colorimetric method. A total of five genes, namely, phoU, pstB, pstA1, pstC, and pstS, were annotated for phosphate solubilization, which is associated with the ability of the Q2H1 strain to solubilize phosphate under in vitro conditions. Results: It is revealed that genes in the Q2H1 genome associated with nitrogen fixation belonged to three groups, namely, nitrogen fixation (nifU, sufU, salA, and nifS), nitrogen metabolism (nirA, nrtB, and nasA), and glutamate synthesis (glnA, gltB, gltD, and gudB), supported by evidence that Q2H1 grew on medium without nitrogen. We have also identified a siderophore gene cluster located on the chromosome of Q2H1, including seven genes (viz., rbsR, rhbf, rhbE, rhbD, rhbC, rhbA, ddc, and an unknown gene). In the in vitro assay, a prominent brown circle around the colony was produced on the chrome azurol S medium at 48 and 72 h post-inoculation, indicating that the siderophore gene cluster in Q2H1 harbored the ability to produce siderophores. Conclusion: In summary, these findings implied that identifying strain-specific genes for their metabolic pathways in bacterial endophytes may reveal a variety of significant functions of plant growth-promoting mechanisms.

The Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant.

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host-pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.

An efficient gene disruption method for the woody plant pathogen Botryosphaeria dothidea.

BACKGROUND: Botryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea. RESULTS: We established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 106 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 µg per 106 protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red. CONCLUSION: The established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.

Occurrence and Distribution of Rhinoncus sibiricus (Coleoptera: Curculionoidea) and Its Preference for Two Buckwheat Species in China.

Rhinoncus sibiricus Faust, a major pest of buckwheat, has invaded the buckwheat cultivation areas of China for years. This pest was first found in Russia in 1940, causing great damage during the entire buckwheat-growing season. In China, there are few records on R. sibiricus, and studies regarding pest damage on buckwheat are unknown. The occurrence and distribution of this species in China is still not clear. We therefore conducted field surveys for 6 yr to identify the distribution range and the degree of pest damage caused by R. sibiricus in the buckwheat-planting areas of China and tested its preference for two Fagopyrum species in common garden experiments. The results showed that R. sibiricus had a larger distribution range in the Northern rather than the Southern part of China, and that pest damage was more serious in northern China. The pest preferred F. tataricum (Tartary buckwheat, Polygonales: Polygonaceae) over F. esculentum (Common buckwheat, Polygonales: Polygonaceae), but caused damage to both, indicating its potential for distribution in southern China. This study clarified the occurrence, distribution, and damage traits of R. sibiricus in the buckwheat cultivation areas of China, which will help explain the pest attack traits and inform strategies for pest control and prediction.