Soft Rot Research Articles

Intracenotic patterns in damage of Abies sibirica Ledeb.-dominated stands by infectious diseases in the south of Central Siberia

Abies sibirica Ledeb.-dominated stands degradation is of special concern. Nevertheless, insufficient attention is paid to the role of pathogens in this process. The aim of the present study is to assess the coenotic role of infectious diseases in fir-dominated stands in the south of Central Siberia. The research was based on a forest pathological examination conducted in A. sibirica-dominated forest stands in the Krasnoyarsk Krai. The cenotic role of the identified diseases is unequal. Stem decay and fir broom rust are typical diseases that weaken trees and induce rotten windsnap accumulation. Fir broom rust randomly affects trees of different age and size. The prevalence of fir broom rust in the studied fir stands reached 10% or more. The most hazard diseases in the studied stands are bacterial soft rot and root rot. Bacterial soft rot damages up to 20% of trees and causes their gradual dieback. The main cause of tree mortality in fir-dominated stands is root rot. The root rot-causing fungi lead to rapid dieback of trees of different cenotic status. In synergy with other factors, root rot acts as a significant endogenous factor in the current reorganization of coniferous forest ecosystems in the south of Central Siberia.

Enhancing shelf-life of carrot (Daucus carota L.) by different treatments during post-harvest processing

The study was carried out on the effect of physical, chemical and biological treatments on carrot (Daucus carota L.) for enhancing shelf-life during post-harvest processing at Department . of Food Process Engineering., Agricultural Engieering college & Research Institute, Tamil Nadu Agricultural University, Coimbatore. The most significant changes in post-harvest quality were weight loss, bitterness, bacterial deterioration, rooting and sprouting. Losses in carrots are due to Sclerotinia rot, Botrytis rot, Bacterial soft rot (Erwinia sp.) and sour rot (Geotrichum sp.). The physical, chemical, biological and combination of these methods were studied. Among the different methods, combination of chlorination and ozonization during washing followed by application of Lactobacillus plantarum 020 at the rate of 10 ml / litre of wash water was more effective. Carrots processed with the treatment had a shelf-life of six days under normal room temperature whereas shelf-life of untreated carrots was two days under normal room temperature.

Poda para curar la pudrición en pitahaya

Abstract This factsheet for farmers in Nicaragua provides information on bacterial soft rot disease on dragon fruit ( Hylocereus spp.) caused by bacteria. It includes details about its symptoms, biology, control and a photo of the affected plant being cut off. Instituto UPOLI /ICIDRI, UNICAM

Pythium spp. – rosnący problem w uprawie ogórka pod osłonami

In 2022 and 2023, a record number of cucumber samples came to the Plant Diseases Clinic in Institute of Plant Protection – National Research Institute in Poznan. The dominant problem (more than 90% of the samples) was the sudden wilting of healthy-looking plants, despite adequate water availability. The yield decreased drastically, and the plants in the entire rows dried up and died prematurely. Until 2019, similar symptoms appeared sporadically on cucumbers delivered to the Clinic. On the basis of microscopic observation, confirmed by isolation on artificial substrates from fragments of infected cucumber, the cause of the disease was identified – fungus-like organisms (Oomycetes) of the genus Pythium. In soilless cultivation systems, they cause soft rot of the roots and the shoot base of cucumber and the so-called sudden wilting of a cucumber. Typical symptoms of infection are stunted plant growth, brown discoloration of the root tips and wilting of the plant during the warmer times of the day (they regain turgor during the night). Infected plants turn yellow and die. Infection can occur all year round. In summer, thermophilic species are the most serious problem, while in winter and early spring, spe¬cies preferring lower temperatures. The samples delivered to the Clinic were dominated by the species Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish commonly known as Pythium ultimum Trow. The importance of Pythium spp. in the cultivation of cucumber under cover increases year by year, taking into account the frequency of occurrence in the samples delivered to the Clinic.

IN VITRO EFFICACY OF DISPARATE FUNGICIDES AGAINST Lasiodiplodia theobromae ROOT ROTS OF ORANGE-FLESHED SWEET-POTATO VARIETIES

Sweet-potato cultivation is limited globally by many storage rots caused mainly by fungi. The study aimed to test the effectiveness of several types of pesticides against <em>Lasioderma theobromae</em> <em>in vitro</em>. Three trials were set up using a completely randomized design with three replications. The first experiment used four isolates of <em>Trichoderma harzianum</em> (AIM16, NSBM, BGMZ4, and AIM3). The second trial was evaluating types of synthetic fungicides, i.e., mancozeb (50%, 100%), mancozeb+carbendazim (50%, 100%), and mancozeb+metalaxyl+Cu (50%, 100%). The third experiment testing botanical pesticides, namely leaf extracts of <em>Ixora, Guieria</em>, and <em>Bauhinia</em>, as well as <em>Eucalyptus</em> resin and <em>Ricinus </em>soap. The four isolates of <em>T. harzianum</em> inhibited the mycelia growth of <em>L. theobromae</em> by 8.0–100%, significantly different from the control, but there was no significant difference between the isolates. Solutions of mancozeb (100%) and mancozeb+carbendazim (50%, 100%) were more effective in suppressing the development of pathogenic mycelia compared to mancozeb+metalaxyl+Cu. Mancozeb treatment inhibited 8.0–100% of pathogen growth and was significantly different compared to other treatments. Plant extracts inhibited the growth of pathogens by 8.0–100%. <em>Ricinus</em> soap (50%, 100%), <em>Eucalyptus</em> (100%), and <em>Guieria</em> (100%) consistently inhibited the growth of pathogenic mycelia by 70%. Plant extracts and mancozeb were effective in suppressing the growth of pathogenic mycelia. The study indicates that <em>T. harziaum</em>, plant extracts, and mancozeb could be developed as a fungicide to control the soft rot fungus <em>L. theobromae</em> of sweet potato tubers

Analysis of the volatile profiles of kiwifruits experiencing soft rot using E-nose and HS-SPME/GC–MS

Soft rot is a postharvest storage disease occurring in kiwifruit that can cause substantial losses and is difficult to detect. In this study, the internal quality of kiwifruits was measured. In addition, the volatile profiles of kiwifruits were analysed by electronic nose (E-nose) and headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS–SPME/GC–MS). The results showed that the firmness, titratable acidity (TA) and ascorbic acid (AA) decreased as the rot worsened, while malondialdehyde increased. A principal component analysis (PCA) of the E-nose data showed a clear distinction between the intact and diseased fruits. A loading analysis (LOA) of the E-nose data showed that the sensor response appeared to significantly vary between the intact and diseased fruits. Nine differential metabolites for the intact and diseased kiwifruits were acquired by HS–SPME/GC–MS, including isobutyl butyrate, ethyl 2-furoate, ethyl valerianate, propyl butyrate, ethyl hexanoate, 4-terpene alcohols, benzyl alcohol, oxime-methoxy-phenyl and 4-isopropyl toluene. There was a significant correlation (p < 0.05) between the physicochemical indices and volatile profiles obtained by E-nose and HS-SPME/GC–MS. This study proved that the combination of E-nose and GC–MS could potentially be used for detecting kiwifruits experiencing soft rot.

First report of bacterial soft rot caused by Enterobacter mori affecting host watermelon.

InJune2021,bacterial stem rot-likesymptomswere observed on the stems and leaves of watermelon (Citrullus lanatus cv. 'Zaojia') in Pingyu County, Zhumadian City, Henan Province, China (32.44N 114.24E), which showed brown to dark brown lesions on the stems (Fig. 1A). The stems then became scorched, and the leaves showed necrotic lesions with small water-soaked spots (Fig. 1B). Watermelon is a very important economic plant in this small county, where the watermelon planting area accounts for about 15% of the arable land area. Approximately 2 hectares of 'Zaojia' have been investigated, and the disease incidence rates were almost 20~30%, thus, causing severe economic losses. Ten symptomatic watermelon stems and leaves were randomly collected based on the typical symptoms, brought into the Lab and used to isolate the pathogen. Each infected tissue was excised and cut into small pieces (about 5 mm×5 mm) and surface disinfected with 1% NaClO for 3 min. The pieces were then rinsed three times in sterile distilled water (SDW) and dried by airing. These pieces (4-5 pieces per sample) were macerated in 200 μL SDW for 60s in a sterile mortar and pestle. A volume of 5 μL suspensions of each sample were streaked onto two LB agar plates and incubated for 48h at 28°C in the dark. After incubation, the colonies on LB agar plate were small, round, raised, white to cream-colored, and had smooth margins (Fig. 2). Two strains from each plate were selected. The genomic DNA of all 40 strains was extracted using a Bacterial Genomic DNA Extraction Kit D1600 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene (27F:5'-AGA GTT TGA TCC TGG CTC AG-3', /1492R: 5'-CTA CGG CTA CCT TGT TAC GA-3'), and the three housekeeping genes, including gyrB (Trantas et al., 2013), icdA and proA (Ma et al., 2007), were amplified. Sequence analysis showed that 40 strains shared the same sequence, so only one sequence was submitted into GenBanK.The 16s rDNA partial sequences (SUB12134746) shared 100% similarity with E.mori (CP084692.1), and the gyrB (OP676246), icdA (OP676248) and proA (OP676247) genes shared 98.67%, 99.39% and 97.99% homology with those of E. mori (CP084692.1), respectively. Besides, the phylogenetic tree analysis based on multi-housekeeping gene joint gryB-icdA-proA showed that E.mori（OP676246-OP676248- OP676247）from watermelon was culsterd with the E.mori (CP084692.1) from South Korea and E.mori (CP055276.1) from kiwifruit (Fig. 3). Thus, E.moriwas confirmed to be the pathogen responsible for bacterial soft rot of watermelon in this study. To confirm the pathogenicity, 15-day-old healthy cv. 'Zaojia' watermelon seedlings were inoculated by spraying all the seedlings with a bacterial suspension (1×10 8 CFU mL-1) at an incubation temperature of 28 °C and 70% relative humidity, and sterile distilled liquid LB medium was applied as a negative control treatment. Three times were conducted for the isolate, and each time included nine watermelon plants. After 10 days, only the inoculated cotyledons and leaves with the bacterial suspension showed bacterial leaf spots that resembled those observed on naturally infected watermelon cotyledons and leaves (Fig. 4A-C), whereas the control plants remained asymptomatic (Fig. 4D). Simultaneously, the watermelon stems were inoculated with the bacterium in vitro. Each stem was slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, and the experiment was repeated three times. Water-soaked symptoms were visible on the stems (Fig. 4E), while the control plants remained asymptomatic (Fig. 4F). The strains were then successfully re-isolated and identified by sequence analyses of their 16S ribosomal RNA gene and gyrB, icdA and proA genes. Therefore, the inoculation experiment of the isolatedbacterium fulfilled Koch's postulates. Previously, E. mori has been reported to cause bacterial wilt on white mulberry (Morus alba L.) (Zhu et al. 2022), peach fruit (Prunus persica) (Ahmad et al. 2021) and kiwifruit (Actinidia deliciosa [A. Chev.] CF Liang et AR Ferguson) (Zhang et al. 2021). To our knowledge, this is the first report of E. mori causing bacterial soft rot on watermelon in world.

Dismantling and reorganizingPseudomonas marginalissensu lato

AbstractUntil recently, it had been a common practice in the field of plant disease control to identify bacterial strains with the following characteristics asPseudomonas marginalis: fluorescent; oxidase‐, potato soft rot‐ and arginine dihydrolase‐positive; soft rot‐causing (here referred to asP.marginalissensu lato). In contrast, as phylogenetic and genomic analyses have been introduced into the bacterial taxonomy, it became clear thatP.marginalissensu lato is heterogeneous, suggesting that it might be a species complex containing many cryptic species. Therefore, a preliminary survey was performed targeting theP.marginalissensu lato strains preserved in the NARO (National Agriculture and Food Research Organization, Japan) Genebank to sort these into strains corresponding toP.marginalissensu stricto (i.e., strains that would be included in the same species range as the type strain ofP.marginalis) and strains that might not fit this category. Since then, attempts have been made to examine the taxonomic affiliations of the latter by using a polyphasic approach. As a result, some strains were proven to differ from any known species and are described as the following novel species:Pseudomonas kitaguniensis,Pseudomonas allii,Pseudomonas cyclaminis,Pseudomonas petroselini,Pseudomonas brassicae,Pseudomonas lactucae,Pseudomonas aegrilactucaeandPseudomonas morbosilactucae. In this article, the profiles of each of these novel species and a scheme for their identification are briefly introduced. In addition to the problem of cryptic species, reorganizingP.marginalissensu lato still faces various challenges, which we discuss.

Manuka Honey Reduces the Virulence of Pectobacterium brasiliense by Suppressing Genes That Encode Plant Cell Wall-Degrading Enzymes

The primary virulence of soft rot pectobacteria, including Pectobacterium brasiliense, is mainly determined by the massive production of plant cell wall-degrading enzymes (PCWDEs), which promote plant tissue maceration in many crops. The antibiotic treatment to treat bacterial soft rot causes environmental problems and potentially affects resistance. Antibiotic resistance is driving interest in antimicrobial treatments, and no organism has been reported to have acquired resistance to honey. However, the use of honey as a therapeutic agent for plant bacterial pathogens has rarely been studied. Therefore, this study was undertaken to determine the in vitro effect of Manuka honey against P. brasiliense at the phenotypic and genotypic levels. A sublethal concentration of honey was determined by a growth inhibition assay in broth medium containing different concentrations of Manuka honey. A macerating assay was performed on orchid leaves, and the activities of the PCWDEs were examined in plate assays. The expression of PCWDE-associated genes was investigated using semi-quantitative reverse transcription-polymerase chain reaction analysis. The exposure of P. brasiliense to a sublethal concentration of Manuka honey significantly decreased the maceration ability of the orchid and the synthesis of PCWDEs, i.e., pectate lyase, polygalacturonase, and protease. Moreover, five PCWDEs-encoding genes, such as pelA, pelB, pelC, pehA, and prtW, had lower expression levels after the honey treatment compared with recA as the internal standard. The honey treatment decreased the virulence of P. brasiliense associated with the synthesis of PCWDEs. Therefore, Manuka honey reduced virulence by suppressing the expression of the PCWDE genes.

First report of a new bacterial stem rot disease of strawberry (Fragaria×ananassa) caused by Pantoea ananatis in Jiangsu, China.

Strawberry (Fragaria×ananassa Duch.) is an important economic fruit crop in the world. With the continuous expansion of strawberry planting area, strawberry disease is one of the most important limiting factors, which seriously affects the agronomic performance and leads to significant economic losses. In November 2020, an infected stem rot disease of strawberries was detected in the strawberry growing area of Donghai County, Jiangsu Province, China. The disease incidence ranged from 30 % to 45 %. Initially, infected plants included stunted growth of new leaves, leaflet asymmetry, and holes in the vertical section of the stem, resulting in leaf blight and death in severe cases. To isolate the pathogen, two symptomatic plants were randomly collected. And then infected plants were surface sterilized with 75 % ethanol for 1 min, followed by 2 % sodium hypochlorite for 6 minutes. After that, the infected plants were washed 4-5 times with double sterilized distilled water, cut into 3-5 mm small pieces, and soaked in 2 ml of sterile water for 15 min, after which 100 µl of liquid suspension were spread onto Luria-Bertani medium (LB) and incubated at 28 °C for 12-16 h. All isolates showed yellow, viscous, round, and smooth (Figure S1, C) and the isolates were designated as JX1 and JX2. To identify the pathogen, the genomic DNA were extracted from isolates using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) and the fragments of gyrB, rpoB and leuS gene were amplified using the primer pairs UP-1S/UP-2Sr (Yamamoto and Harayama 1995), rpoB-F/rpoB-R and leuS-F/leuS-R (Yu et al. 2022), respectively. Sequence analyses showed that the nucleotide sequences of gyrB, rpoB, and leuS fragments of the isolates shared 99.72 %, 99.67 % and 98.37 % identity with the Pantoea ananatis type strain LMG 2665 (KF482590.1, EF988972.1 and KF482626.1, respectively ), which suggests that the isolate could be Pantoea ananatis. To further verify that P. ananatis was identity of these isolates, the whole genome was sequenced using PacBio sequel II technology. The Average Nucleotide Identity (ANI) calculation showed that the whole-genome sequence was 99.0% similar to that of the Pantoea ananatis type strain LMG 2665 (Jain et al. 2018). The isolates were therefore recognized as P. ananatis. To confirm pathogenicity, roots of strawberry plants were inoculated by wounding as described (Wang et al. 2017) with bacterial suspensions (108 CFU/ml) for 30 min, and transplanted into 10 cm ×8.5 cm pots filled with substrate (peat: perlite: vermiculite =3:1:1). The negative control plants were inoculated with sterile distilled water (20 individual plants per group). All infected plants were placed in a greenhouse under the following environmental conditions: 30 ℃/25 ℃ day/night, >70 % relative humidity, 16-h/8-h photoperiod. The experiment was carried out three times. After 3 to 4 weeks of inoculation, the new leaves of the plants were smaller and asymmetrical, while the negative plants remained healthy. After 8 weeks, a significant stem rot pocket developed on all inoculated plants, similar to the symptoms observed in the field. In contrast, no symptoms were observed in negative plants (Figure S2). To fulfill Koch's postulates bacteria were further isolated, purified and identified from the greenhouse inoculated plants. The results proved that the causative agent of strawberry stem rot was P. ananatis. In recent decades, P. ananatis has been found to cause bacterial leaf blight in strawberries (Bajpai et al. 2020). It has also caused other crop diseases, such as maize white spot, peach soft rot and others (Cui et al.2022; Liao et al. 2015). Although other crop diseases caused by P. ananatis, a bacterial pathogen, there has been no report of P. ananatis causing the symptoms of stem rot disease in strawberry. To our knowledge, this is the first report of P. ananatis causing stem rot in strawberry. This study provides solid evidence that strawberry stem rot disease in China can also be caused by the novel pathogen Pantoea ananatis. In conclusion, this report will provide a theoretical reference for the prevention and control measures of P. ananatis causing strawberry stem rot disease in the future.

Phytopathogenic Bacteria Associated with Bacterioses of Common Oak (Quercus robur L.) in Ukraine

Studying the biological characteristics of the causative agents of bacteriosis of the common oak (Quercus robur L.) (to highlight the links between the key factors of pathogenesis and bacterial groups) is a new and relevant area of research. This work aims to identify the species composition of phytopathogenic bacteria associated with common oak bacteriosis and to study the morphological, cultural, and biochemical properties of pathogens. To establish the general phytosanitary state of the surveyed stands, classical forest inventory and phytopathological (visual on based on typical symptoms of the disease and macroscopic) methods were used. The properties of isolated bacteria were studied using microbiological methods. The weakening and dieback of common oak in the forests of Ukraine is directly related to four types of bacterioses and their pathogens: the soft bacterial rot of acorns (Pectobacterium carotovorum subsp. carotovorum, bacterial wetwood (Lelliottia nimipressuralis), dry rot of branches and trunks (Erwinia rhapontici), and canker disease (Pseudomonas fluorescens and Pseudomonas sp.). Research on the correspondence between the biological properties of common oak bacteriosis pathogens has established that the synthesis of pectin-degrading enzymes and the ability to ferment with the release of acid and gas in many carbohydrate media by phytopathogenic bacteria is the main characteristic. This ensures the intensity and typical symptoms of the pathogenesis of a particular bacteriosis.

Natural Infections of Potato Plants Grown from Minitubers with Blackleg-Causing Soft Rot Pectobacteriaceae.

Information on the infection incidence of blackleg-causing soft rot Pectobacteriaceae (BL-SRP) in potato crops grown from minitubers (PB1-crop) and the distribution of BL-SRP in individual plants was collected during a two-year survey conducted at five potato growers located in the Netherlands. In the last weeks before haulm destruction, leaves, stems, and tubers of 100 or 200 plants were analyzed separately for the presence of Pectobacterium parmentieri, P. brasiliense, P. atrosepticum, and Dickeya spp. Extracted plant parts enriched for BL-SRP were analyzed with TaqMan assays specific for the detection of blackleg-causing BL-SRP. In 2019, low incidences of P. parmentieri (1-6%) in leaves were found at four growing sites. At one farm, reactions were detected in TaqMan assays for D. zeae and D. chrysanthemi in leaves. In 2020, the crops of two growers were largely free from BL-SRP. At one farm, a high infection incidence (21%) was found for D. fangzhongdai in tubers. The isolated pathogen was able to cause potato blackleg. At two other farms, high infection incidences in tubers were found with P. brasiliense (35-39%) and P. parmentieri (12-19%), whereas the incidence of P. brasiliense in leaves was also high (8%). In conclusion, high infection incidences with BL-SRP in potatoes can be found in a PB1 crop at the end of the growing season. Infections in individual plants were found either in tubers or in leaves. The potential sources of initial infection are discussed.

Innovative biosynthesis, artificial intelligence-based optimization, and characterization of chitosan nanoparticles by Streptomyces microflavus and their inhibitory potential against Pectobacterium carotovorum

Microbial-based strategy in nanotechnology offers economic, eco-friendly, and biosafety advantages over traditional chemical and physical protocols. The current study describes a novel biosynthesis protocol for chitosan nanoparticles (CNPs), employing a pioneer Streptomyces sp. strain NEAE-83, which exhibited a significant potential for CNPs biosynthesis. It was identified as Streptomyces microflavus strain NEAE-83 based on morphological, and physiological properties as well as the 16S rRNA sequence (GenBank accession number: MG384964). CNPs were characterized by SEM, TEM, EDXS, zeta potential, FTIR, XRD, TGA, and DSC. CNPs biosynthesis was maximized using a mathematical model, face-centered central composite design (CCFCD). The highest yield of CNPs (9.41 mg/mL) was obtained in run no. 27, using an initial pH of 5.5, 1% chitosan, 40 °C, and a 12 h incubation period. Innovatively, the artificial neural network (ANN), was used for validating and predicting CNPs biosynthesis based on the trials data of CCFCD. Despite the high precision degree of both models, ANN was supreme in the prediction of CNPs biosynthesis compared to CCFCD. ANN had a higher prediction efficacy and, lower error values (RMSE, MDA, and SSE). CNPs biosynthesized by Streptomyces microflavus strain NEAE-83 showed in-vitro antibacterial activity against Pectobacterium carotovorum, which causes the potato soft rot. These results suggested its potential application for controlling the destructive potato soft rot diseases. This is the first report on the biosynthesis of CNPs using a newly isolated; Streptomyces microflavus strain NEAE-83 as an eco-friendly approach and optimization of the biosynthesis process by artificial intelligence.

The involvement of the phenylpropanoid and jasmonate pathways in methyl jasmonate-induced soft rot resistance in kiwifruit (Actinidia chinensis).

Botryosphaeria dothidea is a major postharvest causal agent of soft rot in kiwifruit. Methyl jasmonate (MeJA) is an important plant hormone that participates as a plant defense against pathogens from a signal molecule. However, the impact and regulatory mechanism of MeJA on the attenuation of kiwifruit fungal decay remains unknown. This work investigated the effects of exogenous MeJA on the enzyme activity, metabolite content and gene expression of the phenylpropanoid and jasmonate pathways in kiwifruit. The results revealed that MeJA inhibited the expansion of B. dothidea lesion diameter in kiwifruit (Actinidia chinensis cv. 'Hongyang'), enhanced the activity of enzymes (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate: coenzyme A ligase, cinnamyl alcohol dehydrogenase, peroxidase and polyphenol oxidase), and upregulated the expression of related genes (AcPAL, AcC4H, Ac4CL, and AcCAD). The accumulation of metabolites (total phenolics, flavonoids, chlorogenic acid, caffeic acid and lignin) with inhibitory effects on pathogens was promoted. Moreover, MeJA enhanced the expression of AcLOX, AcAOS, AcAOC, AcOPR3, AcJAR1, AcCOI1 and AcMYC2 and reduced the expression of AcJAZ. These results suggest that MeJA could display a better performance in enhancing the resistance of disease in kiwifruit by regulating the phenylpropanoid pathway and jasmonate pathway.

Controlling soft rot of postharvest chilli pepper (Capsicum annuum L.) by an antagonist Bacillus amyloliquefaciens S917: Efficacy and action mode

Pectobacterium carotovorum, the causal agent of soft rot, is responsible for significant economic damage in storage and transportation of postharvest chilli pepper. In this study, an antagonist S917, identified as Bacillus amyloliquefaciens based on the 16S rDNA sequence, was screened from 1400 strains for antagonistic activity towards Pectobacterium carotovorum subsp. brasiliense XC1 (Pcb XC1). The in vitro antibacterial result showed that cells and peptide extract of B. amyloliquefaciens S917 had good inhibitory activity against Pcb XC1. Based on draft genome sequences of B. amyloliquefaciens S917, five bacteriocins (Thusin_alpha, Thusin_beta2, Amylocyclicin, ComX1, LCI-like) were identified by BAGEL4 database and four antibacterial metabolites (Bacillaene, Macrolactin H, Bacilysin, Bacillibactin) were identified by antiSMASH database. The in vivo disease control result of chilli pepper showed that cells of B. amyloliquefaciens S917 performed better in reducing the rotting incidence and lesion diameter. Result of gas chromatography-ion mobility spectrometry (GC-IMS) showed that B. amyloliquefaciens S917 cells (BSC) treatment protected the original volatile aroma of chilli pepper. However, the control had richer volatile compounds related to soft rot, including methylbutanoate, dimethyl sulfide, methylbutanal, 3-methyl-2-butenal, pentan-1-ol, 3-methylbutan-1-ol, hexanol. Further, action mode of BSC was investigated. The number of green fluorescent protein labelled Pcb XC1 in chilli pepper was reduced by more than 99% after BSC treatment. Result of scanning electron microscopy also showed that BSC treatment prevented the damage of pepper epidermal structure caused by Pcb XC1. Moreover, the result of RNA-seq showed that BSC treatment triggered defense responses of chilli pepper. Pathways of plant pathogen interaction, phenylpropanoid biosynthesis and metabolism, plant hormone synthesis and signal transduction, MAPK signaling, WRKY transcription factor, and cell wall synthesis were activated. This study confirmed that B. amyloliquefaciens S917 can be used as an antagonist to control soft rot of postharvest chilli pepper.

Bacillus velezensis (strains A6 & P42) as a potential biocontrol agent against Klebsiella variicola, a new causal agent of soft rot disease in carrot.

Bacterial soft rot is one of the most devastating diseases and a major constraint encountered during carrot farming. Biological agents are the best eco-friendly alternatives to agrochemicals to manage soft rot disease to ensure environmental sustainability. In this study, about eight isolates of bacterial pathogen causing soft rot in carrots were collected from Karnataka, India. Based on the 16S rRNA sequencing the pathogen isolates causing soft rot of carrot were identified as Klebsiella variicola. The morphological characteristics of K. variicola was investigated under scanning electron microscopy. The pathogenicity assay showed that all eight isolates were pathogenic to the carrot. An in vitro and in planta assay of two novel strains of Bacillus velezensis (A6 and P42) against K. variicola indicated that both strains had strong antagonistic activity against all the pathogen strains. Furthermore, the volatile bioactive compounds produced by A6 and P42 strains were analyzed in GC-MS, which revealed the presence of 10 and 6 bioactive compounds in their culture filtrate, respectively, with antibacterial and antifungal properties. The present study suggests that both A6 and P42 strains of B. velezensis were antagonistic to K. variicola and can be used as biocontrol agents to manage soft rot diseases of carrot under field conditions.

Involvement of PpMYB306 in Pichia guilliermondii-induced peach fruit resistance against Rhizopus stolonifer

Antagonistic yeasts are promising agents for inducing host resistance against pathogens in harvested fruit, but the transcriptional regulatory mechanism involved remains unclear. In our study, Pichia guilliermondii treatment increased the enzyme activities and gene expression of cinnamate 4-hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) and maintained a higher lignin content in peach fruit after R. stolonifer infection. Transcriptome analysis revealed a total of 17 differentially expressed genes (DEGs) encoding MYB transcription factors (TFs); among them, the gene expression of PpMYB306 was downregulated by P. guilliermondii treatment. In addition, by binding to the promoters of PpC4H, PpCAD1, and PpPOD44, PpMYB306 negatively regulated lignin biosynthesis in peach fruit. Furthermore, transient overexpression of PpMYB306 in peach fruit decreased the expression of PpC4H, PpCAD1, and PpPOD44, reducing the lignin content and thus increasing the lesion diameter associated with soft rot. Taken together, these findings suggested that P. guilliermondii treatment inhibited PpMYB306-mediated the transcriptional repression of lignin biosynthesis-related genes in peach fruit after R. stolonifer infection, which contributed to maintaining a higher lignin content, thereby improving the disease resistance of peach fruit.

Early warning of Diaporthe infection in kiwifruit soft rot by plasmonic dimer-enhanced Raman spectroscopy.

Diaporthe genus is the dominant pathogens of kiwifruit soft rot with long incubation period and rapid onset and very hard to detect in advance. It is of great significance to develop point-of-care tests for disease prevention and field management. Here we screen a pair of genus-level primers for constructing plasmonic dimer Rayleigh/Raman spectroscopy, which enables rapid, specific, and sensitive detection of Diaporthe genus in real kiwifruits. Dark-filed Rayleigh scattering clearly visualizes the target-induced dimer assembly of nanoprobes. Plasmonic dimer-enhanced Raman spectroscopy has high specificity for 12 common isolates of Diaporthe genus in one batch samples with highly accurate results and much higher sensitivity than polymerase chain reaction (PCR). It realizes the recognition of Diaporthe infection at an early stage of 24h when the kiwifruits do not have any noticeable symptoms. It demonstrates a bright prospect of point-of-care tests for early warning of kiwifruit soft rot infection and quality control.

A New Approach Using the SYBR Green-Based Real-Time PCR Method for Detection of Soft Rot Pectobacterium odoriferum Associated with Kimchi Cabbage.

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferum- specific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation.

Soft rot causes significant economic losses in the kiwifruit industry. This study isolated strain CTXW 7-6-2 from healthy kiwifruit tissue; this was a gram-positive bacterium that produced the red pigment pulcherrimin. The phylogenetic tree based on 16S ribosomal RNA, gyrA, rpoB, and purH gene sequences identified CTXW 7-6-2 as a strain of Bacillus subtilis. CTXW 7-6-2 inhibited hyphal growth of pathogenic fungi that cause kiwifruit soft rot, namely, Botryosphaeria dothidea, Phomopsis sp., and Alternaria alternata, by 81.76, 69.80, and 32.03%, respectively. CTXW 7-6-2 caused the hyphal surface to become swollen and deformed. Volatile compounds (VOC) produced by the strain inhibited the growth of A. alternata and Phomopsis sp. by 65.74 and 54.78%, respectively. Whole-genome sequencing revealed that CTXW 7-6-2 possessed a single circular chromosome of 4,221,676 bp that contained 4,428 protein-coding genes, with a guanine and cytosine (GC) content of 43.41%. Gene functions were annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, Carbohydrate-Active enZYmes, and Rapid Annotations using Subsystem Technology databases, revealing non-ribosomal pathways associated with antifungal mechanisms, biofilm formation, chemotactic motility, VOC 3-hydroxy-2-butanone, cell wall-associated enzymes, and synthesis of various secondary metabolites. antiSMASH analysis predicted that CTXW 7-6-2 can produce the active substances bacillaene, bacillibactin, subtilosin A, bacilysin, and luminmide and has four gene clusters of unknown function. Quantitative real-time PCR (qRT-PCR) analysis verified that yvmC and cypX, key genes involved in the production of pulcherrimin, were highly expressed in CTXW 7-6-2. This study elucidates the mechanism by which B. subtilis strain CTXW 7-6-2 inhibits pathogenic fungi that cause kiwifruit soft rot, suggesting the benefit of further studying its antifungal active substances.