Host plant physiological transformation and microbial population heterogeneity as important determinants of the Soft Rot Pectobacteriaceae-plant interactions.

Pectobacterium and Dickeya species belonging to the Soft Rot Pectobacteriaceae (SRP) are one of the most devastating phytopathogens. They degrade plant tissues by producing an arsenal of plant cell wall degrading enzymes. However, SRP-plant interactions are not restricted to the production of these "brute force" weapons. Additionally, these bacteria apply stealth behavior related to (1) manipulation of the host plant via induction of susceptible responses and (2) formation of heterogeneous populations with functionally specialized cells. Our review aims to summarize current knowledge on SRP-induced plant susceptible responses and on the heterogeneity of SRP populations. The review shows that SRP are capable of adjusting the host's hormonal balance, inducing host-mediated plant cell wall modification, promoting iron assimilation by the host, stimulating the accumulation of reactive oxygen species and host cell death, and activating the synthesis of secondary metabolites that are ineffective in limiting disease progression. By this means, SRP facilitate host plant susceptibility. During host colonization, SRP populations produce various functionally specialized cells adapted for enhanced virulence, increased resistance, motility, vegetative growth, or colonization of the vascular system. This enables SRP to perform self-contradictory tasks, which benefits a population's overall fitness in various environments, including host plants. Such stealthy tactical actions facilitate plant-SRP interactions and disease progression.

Isolation, identification, and pathogenicity of Pseudomonas glycinae causing ginseng bacterial soft rot in China.

By tissue separation method, tie-back experiment, and hypersensitive response test in potato, strain XJFL-1 was isolated and identified as the pathogen of ginseng bacterial soft rot in Liaoning Provence, China. The morphological characteristics of XJFL-1 were conformed to the Pseudomonads genus. Microbial fatty acid identification showed the principal cellular fatty acid traits of XLFJ-1 corresponded with Pseudomonas spp. API 50CH test results allowed the differentiation of strain XJFL-1 and MS586T from other closely related Pseudomonas species. The molecular identification, including 16S rRNA analysis and multilocus sequence typing (MLST) analysis, showed that XJFL-1 was in the same branch as P. glycinae MS586T. The genome of XJFL-1 was 6,296,473 bp, with an average guanine/cytosine (G + C) content of 60.72 %. Comparative genomics analysis using ANIb and GGDC algorithms indicated that the maximum value was observed between XJFL-1 and P. glycinae MS586T. The above morphological, cell morphology, and molecular biological identification results supported to identification of XJFL-1 as P. glycinae. This is the first report of P. glycinae as the plant pathogen causing ginseng bacterial root rot in China, which complements the biological significance of the species to a certain extent, enriches the pathogens of ginseng bacterial soft rot, and provides a theoretical basis for further investigation.

Pectobacterium araliae sp. nov., a pathogen causing bacterial soft rot of Japanese angelica tree in Japan.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA-DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95-96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

Bacteriophage cocktail for biocontrol of soft rot disease caused by Pectobacterium species in Chinese cabbage.

Pectobacterium spp. are necrotrophic plant pathogens that cause the soft rot disease in Chinese cabbage, resulting in severe yield loss. The use of conventional antimicrobial agents, copper-based bactericides, and antibiotics has encountered several limitations, such as bioaccumulation on plants and microbial resistance. Bacteriophages (phages) are considered promising alternative antimicrobial agents against diverse phytopathogens. In this study, we isolated and characterized two virulent phages (phiPccP-2 and phiPccP-3) to develop a phage cocktail. Morphological and genomic analyses revealed that two phages belonged to the Tevenvirinae and Mccorquodalevirinae subfamilies, respectively. The phiPccP-2 and phiPccP-3 phages, which have a broad host range, were stable at various environmental conditions, such as various pHs and temperatures and exposure to ultraviolet light. The phage cocktail developed using these two lytic phages inhibited the emergence of phage-resistant bacteria compared to single-phage treatments in in vitro challenge assays. The phage cocktail treatment effectively prevented the development of soft rot symptom in matured Chinese cabbage leaves. Additionally, the phage cocktail comprising three phages (phiPccP-1, phiPccP-2, and phiPccP-3) showed superior biocontrol efficacy against the mixture of Pectobacterium strains in Chinese cabbage seedlings. These results suggest that developing phage cocktails is an effective approach for biocontrol of soft rot disease caused by Pectobacterium strains in crops compared to single-phage treatments. KEY POINTS: •Two newly isolated Pectobacterium phages, phiPccP-2 and phiPccP-3, infected diverse Pectobacterium species and effectively inhibited the emergence of phage-resistant bacteria. •Genomic and physiological analyses suggested that both phiPccP-2 and phiPccP-3 are lytic phages and that their lytic activities are stable in the environmental conditions under which Chinese cabbage grows. •Treatment using a phage cocktail comprising phiPccP-2 and phiPccP-3 efficiently suppressed soft rot disease in detached mature leaves and seedlings of Chinese cabbage, indicating the applicability of the phage cocktail as an alternative antimicrobial agent.

Lycium barbarum Is a New Host of Botryosphaeria dothidea Associated with Soft Rot Disease in China.

Goji berry (Lycium barbarum) is a plant of the Solanaceae family that is cultivated in the Chinese provinces of Xinjiang, Ningxia, Gansu, and Qinghai, and its fruit is used as a traditional Chinese medicine (Yossa Nzeuwa et al. 2019). In July 2019, fruit rot was observed at an incidence of 20 to 25% on the Goji berry at a fruit market in Yinchuan, Ningxia, China. The fruit symptoms began as slightly shriveled areas on fruit peel, with noticeable softening of the infested portion of the tissue, followed by rotting and a sour odor. To isolate the pathogen, ten symptomatic tissues were randomly collected from different boxes, surface-sterilized for 30 s with 75% ethanol, followed by 0.1% mercuric chloride, then rinsed in sterile distilled water three times and plated onto PDA. The plates were incubated at 25°C in the dark for 7 days. Five purified fungal isolates from different fruit were obtained and single-spores. Emergent fungal colonies were white with 1 to 3 mm white margins and abundant aerial hyphae, 1 to 6 mm high, that became dark gray after 4 to 5 days. Conidia were hyaline, unicellular, fusiform, and measured 19.3 to 28.2 µm× 3.8 to 6.4 µm (n=50). All the morphological characteristics were consistent with Botryosphaeria spp. (Slippers et al. 2004). Five representative isolates, BJN1-BJN5, were selected for molecular identification. Total genomic DNA of the isolates was extracted with a Plant/Fungi DNA Isolation Kit. Translation elongation factor 1-alpha (EF1) gene and internal transcribed spacer (ITS) regions were amplified with primers EF1-728F/986R (Carbone and Kohn 1999) and ITS1/ITS4, respectively. The sequencing results of the five isolates were consistent, and those of the isolate BJN1 we deposited in the NCBI GeneBank database for EF1 (MK733274) and ITS (MK359291). A BLAST search of the GenBank database indicated that the EF1 and ITS sequences had 100% and 99% similarity, respectively, to B. dothidea ex-type strain (AY236898 and KF766151). A phylogenetic tree was constructed using maximum parsimony methods in MEGA11 and BJN1 isolate clustered with the reference sequence of B. dothidea. Pathogenicity tests were performed, inoculating healthy fruit with both mycelial plugs (7 days old) and conidial suspension (1 × 106 conidia/ml), repeated three times. Mycelial plugs of five isolates (BJN1-BJN5) growing on PDA with a colony diameter of 4 mm were placed on the sterilized surface of 20 Goji berry fruit. Sterile PDA plugs were placed on 12 healthy fruit as a control. In a second test, conidial suspensions of five isolates were sprayed on the surface of 20 healthy fruit and sterilized distilled water was used as a control. The inoculated fruits were maintained in an artificial climate chamber at 25°C and 80% to 85% relative humidity with a 12-h photoperiod for 7 days. The development of soft rot, similar to that observed on the original samples, was observed on inoculated fruit while control fruits remained asymptomatic. The pathogen was reisolated from infected fruit and confirmed as B. dothidea based on morphological characteristics and molecular sequences. To our knowledge, this is the first report of B. dothidea causing postharvest fruit rot of Goji berry, and this pathogen has been reported to cause fruit rot in Kiwifruit (Li et al. 2016) and Yellowhorn (Liu et al. 2018). This study provides information on a new postharvest fruit rot of Goji berry in China that has the potential to cause economic losses.

First report of Pectobacterium brasiliense causing blackleg and soft rot of potato in Argentina.

Blackleg and soft rot diseases represent a major threat to the health of potato (Solanum tuberosum) and other vegetable, ornamental and fruit crops worldwide; their main causal agents are species of Pectobacterium and Dickeya. In May 2022, 60% of potato plants (cv. Spunta) in a production field in Córdoba, Argentina (31°32'36''S 64°09'46''W) showed soft rot, blackleg and wilt. To isolate the causal agent, decayed plant tissues were disinfected in 2% NaClO, macerated in sterile water and streaked on crystal violet pectate (CVP) medium. Plates were incubated at 28°C for 48 h. Colonies that produced a pit on CVP medium were purified on nutrient agar. Two of the isolates, called 1Aia and 1B, were characterized by tests commonly employed for the identification of pectinolytic bacteria (Schaad et al. 2001). Both produced Gram-negative rods that were facultatively anaerobic, oxidase negative, nonfluorescent on King´s B, resistant to erythromycin and caused soft rot of potato slices. In addition, these isolates did not produce the blue pigment indigoidine and grew on nutrient glucose agar containing 5% NaCl. Phenotypic characteristics of the isolates 1Aia and 1B were compatible with Pectobacterium spp. Genomic DNA was extracted using the commercially available Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions for the purification of DNA from Gram-negative bacteria. The isolates were positive in a PCR assay for Pectobacterium brasiliense (Duarte et al. 2004). The purified DNA of isolate 1Aia was used to construct a pooled Illumina library, which was sequenced at the Genomics Unit from the National Institute of Agricultural Technology (INTA, Argentina), by using high-throughput Illumina sequencing technology. Average nucleotide identity (ANI) calculation performed by FastANI v0.1.3 (Jain et al. 2018) showed 96.11% identity between the genome of the type strain LMG 21371 of P. brasiliense (Acc. no. JQOE00000000) and our strain 1Aia (Acc. no. JAYGXQ000000000). For pathogenicity test, 3-weeks-old potato plants (cv. Spunta) planted in pots were infiltrated with 10 µl of a bacterial suspension (1x107 CFU/ml) 5 cm above the base of the stem using a sterile syringe. Negative controls were infiltrated with sterile water. Plants were kept under greenhouse conditions and regularly watered. The experiment was performed twice with six plants per treatment. Two days after inoculation, plants treated with P. brasiliense strain 1Aia or 1B showed necrotic lesions on the stems and tubers soft rot symptoms while control plants remained asymptomatic. To fulfill Koch´s postulates, bacteria were re-isolated from symptomatic plants. Re-isolated bacteria, called 1Aia d and 1B d, were confirmed as P. brasiliense according to biochemical and PCR results, as outlined above. Also, the % ANI value between P. brasiliense isolates 1Aia and 1Aia d was 99.99% (Acc. no. JAYGXR000000000). To our knowledge, this is the first report of the occurrence of P. brasiliense in Argentina. This pathogen has been observed causing blackleg and tuber soft rot on potato in Brazil (Duarte et al. 2004), Netherlands (Nunes Leite et al. 2014), Switzerland (de Werra et al. 2015), Russia (Voronina et al. 2019), Serbia (Loc et al. 2022) and USA (Zhang et al. 2023), among other countries worldwide. Due to the important economic and nutritional value of the crop, the distribution of P. brasiliense needs to be investigated and monitored in order to develop effective control strategies.

First report of Lasiodiplodia pseudotheobromae causing soft rot of plum in China.

Plum (Prunus salicina) is one of the most important fruit tree species worldwide (Valderrama-Soto et al. 2021). In June 2023, the postharvest soft rot symptoms were observed on plum fruits in several fruit markets of Guiyang city, Guizhou province, China. The disease incidence in these markets ranged from 20 to 25% with 70% disease severity. Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. In severe conditions, whole fruits become rotted and were covered with white fungal mycelia. Small sections (5 × 3 mm) from 6 diseased plum fruits were surface sterilized by using 75% ethanol for 30 s followed by 0.1% mercuric chloride solution for 5 min, rinsed three times with ddH2O, and then transferred onto potato dextrose agar (PDA) and incubated at 25 ± 2°C for three days. Three pure cultures (GUCC23-0001 to GUCC23-0003) were obtained by transferring a single hyphal tip to new PDA plates. Colonies of these isolates were grayish-white initially, gradually turning to whitish brown with fluffy aerial mycelia and uneven edges and finally turned to a dark gray colony after five days of inoculation. The pseudoparaphyses were hyaline, cylindrical, aseptate, and rounded at apex. Conidia were ellipsoidal, hyaline, unicellular, and 24.2 to 28.6 × 12.3 to 15.5 µm in size (n = 30) (Fig. S1), which were similar to the morphology of Lasiodiplodia pseudotheobromae (Alves et al. 2008). Furthermore, fungal DNA was extracted from fresh mycelia of PDA after seven days by using fungus genomic DNA extraction kit (Biomiga, USA). Partial DNA sequences from four loci including internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and polymerase II second largest subunit (rpb2) were amplified with ITS1 and ITS4 (White et al. 1990), EF1-688F and EF1-1251R (Alves et al. 2008), Bt2a and Bt2b (Glass and Donaldson 1995), and RPB2-LasF and RPB2-LasR, respectively (Cruywagen et al. 2017). GenBank accession numbers are OR361680, OR361681, OR361682 for ITS, OR423394, OR423395, OR423396 for tef1, OR423397, OR423398, OR423399 for tub2, and OR423391, OR423392, OR423393 for rpb2, and gene sequencing showed 99.6 to 100% identity with ex-type strain of L. pseudotheobromae (CBS 116459). Phylogenetic analysis also placed our isolates in a highly supported clade with the reference isolate of L. pseudotheobromae (Fig. S2). Another experiment was designed to confirm the pathogenicity test for additional confirmation. Five mm mycelial plugs of L. pseudotheobromae from a three day old culture on PDA were placed on five surface-sterilized and non-wounded plum fruits for 12 hours and incubated at 25°C ± 2°C for four days. Sterilized fungus free PDA plugs were used as a negative control. Mycelial plugs were removed after 12 hours following which whole fruits were incubated in plastic boxes at 25°C ± 2°C. The experiment was repeated twice. The pathogenicity was evaluated under control conditions in laboratory (relative humidity, 70 ± 5% and temperature 25 ± 5˚C). Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. These symptoms and signs were similar to the initially observed symptoms on plums in the markets. No disease symptoms were observed on the control fruits. The re-isolated fungus obtained from inoculated plum fruits was very similar to those isolated from diseased samples in morphology, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of L. pseudotheobromae causing postharvest fruit rot of plum in China. In 2022, the total planting area of plum was 1946.5 thousand hectares, which produces approximately 6626300 tons of plum (Food and Agriculture Organization of the United Nations, 2022). Based on the disease incidence and severity reported in the current study, soft rot of plum may be responsible for nearly 35% of yield losses under severe. Therefore, our study laid a theoretical foundation for the prevention and control of this post-harvest disease of plum.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

Plant growth-promoting and biocontrol traits of endophytic Bacillus licheniformis against soft rot causing Pythium myriotylum in ginger plant.

Bacterial endophytes from plants harbor diverse metabolites that play major roles in biocontrol and improve plant growth. In this study, a total of 12 endophytic bacteria were isolated from the ginger rhizome. The strain K3 was highly effective in preventing mycelia growth of Pythium myriotylum (78.5 ± 1.5% inhibition) in dual culture. The cell-free extract (2.5%) of endophyte K3 inhibited 76.3 ± 4.8% mycelia growth, and 92.4 ± 4.2% inhibition was observed at a 5% sample concentration. The secondary metabolites produced by Bacillus licheniformis K3 showed maximum activity against Pseudomonas syringae (24 ± 1 mm zone of inhibition) and Xanthomonas campestris (28 ± 3 mm zone of inhibition). The strain K3 produced 28.3 ± 1.7 IU mL-1 protease, 28.3 ± 1.7 IU mL-1 cellulase, and 2.04 ± 0.13 IU mL-1 chitinase, respectively. The ginger rhizome treated with K3 in the greenhouse registered 53.8 ± 1.4% soft rot incidence, and the streptomycin-treated pot registered 78.3 ± 1.7% disease incidence. The selected endophyte K3 improved ascorbate peroxidase (1.37 ± 0.009 µmole ASC min-1 mg-1 protein), catalase (8.7 ± 0.28 µmole min-1 mg-1 protein), and phenylalanine ammonia-lyase (26.2 ± 0.99 Umg-1) in the greenhouse. In addition, K3 treatment in the field trial improved rhizome yield (730 ± 18.4 g) after 180 days (p < 0.01). The shoot length was 46 ± 8.3 cm in K3-treated plants, and it was about 31% higher than the control treatment (p < 0.01). The lytic enzyme-producing and growth-promoting endophyte is useful in sustainable crop production through the management of biotic stress.

Looking for Resistance to Soft Rot Disease of Potatoes Facing Environmental Hypoxia.

Plants are exposed to various stressors, including pathogens, requiring specific environmental conditions to provoke/induce plant disease. This phenomenon is called the "disease triangle" and is directly connected with a particular plant-pathogen interaction. Only a virulent pathogen interacting with a susceptible plant cultivar will lead to disease under specific environmental conditions. This may seem difficult to accomplish, but soft rot Pectobacteriaceae (SRPs) is a group virulent of pathogenic bacteria with a broad host range. Additionally, waterlogging (and, resulting from it, hypoxia), which is becoming a frequent problem in farming, is a favoring condition for this group of pathogens. Waterlogging by itself is an important source of abiotic stress for plants due to lowered gas exchange. Therefore, plants have evolved an ethylene-based system for hypoxia sensing. Plant response is coordinated by hormonal changes which induce metabolic and physiological adjustment to the environmental conditions. Wetland species such as rice (Oryza sativa L.), and bittersweet nightshade (Solanum dulcamara L.) have developed adaptations enabling them to withstand prolonged periods of decreased oxygen availability. On the other hand, potato (Solanum tuberosum L.), although able to sense and response to hypoxia, is sensitive to this environmental stress. This situation is exploited by SRPs which in response to hypoxia induce the production of virulence factors with the use of cyclic diguanylate (c-di-GMP). Potato tubers in turn reduce their defenses to preserve energy to prevent the negative effects of reactive oxygen species and acidification, making them prone to soft rot disease. To reduce the losses caused by the soft rot disease we need sensitive and reliable methods for the detection of the pathogens, to isolate infected plant material. However, due to the high prevalence of SRPs in the environment, we also need to create new potato varieties more resistant to the disease. To reach that goal, we can look to wild potatoes and other Solanum species for mechanisms of resistance to waterlogging. Potato resistance can also be aided by beneficial microorganisms which can induce the plant's natural defenses to bacterial infections but also waterlogging. However, most of the known plant-beneficial microorganisms suffer from hypoxia and can be outcompeted by plant pathogens. Therefore, it is important to look for microorganisms that can withstand hypoxia or alleviate its effects on the plant, e.g., by improving soil structure. Therefore, this review aims to present crucial elements of potato response to hypoxia and SRP infection and future outlooks for the prevention of soft rot disease considering the influence of environmental conditions.

A Pythium myriotylum Small Cysteine-Rich Protein Triggers Immune Responses in Diverse Plant Hosts.

The oomycete Pythium myriotylum is a necrotrophic pathogen that infects many crop species worldwide, including ginger, soybean, tomato, and tobacco. Here, we identified a P. myriotylum small cysteine-rich protein, PmSCR1, that induces cell death in Nicotiana benthamiana by screening small, secreted proteins that were induced during infection of ginger and did not have a predicted function at the time of selection. Orthologs of PmSCR1 were found in other Pythium species, but these did not have cell death-inducing activity in N. benthamiana. PmSCR1 encodes a protein containing an auxiliary activity 17 family domain and triggers multiple immune responses in host plants. The elicitor function of PmSCR1 appears to be independent of enzymatic activity, because the heat inactivation of PmSCR1 protein did not affect PmSCR1-induced cell death or other defense responses. The elicitor function of PmSCR1 was also independent of BAK1 and SOBIR1. Furthermore, a small region of the protein, PmSCR186-211, is sufficient for inducing cell death. A pretreatment using the full-length PmSCR1 protein promoted the resistance of soybean and N. benthamiana to Phytophthora sojae and Phytophthora capsici infection, respectively. These results reveal that PmSCR1 is a novel elicitor from P. myriotylum, which exhibits plant immunity-inducing activity in multiple host plants. [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.

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.

Antifungal activity and mechanism of tetramycin against Alternaria alternata, the soft rot causing fungi in kiwifruit.

Kiwifruit rot caused by the fungus Alternaria alternata occurs in many countries, leading to considerable losses during kiwifruit production. In this study, we evaluated the antifungal activity and mechanism of tetramycin against kiwifruit soft rot caused by Alternaria alternata. Tetramycin exerted antifungal effects through the suppression of mycelial growth, conidial germination, and the pathogenicity of A. alternata. Scanning electron microscopic observations revealed that tetramycin destroyed the mycelial structure, causing the mycelia to twist, shrink, and even break. Furthermore, transmission electron microscopy revealed that tetramycin caused severe plasmolysis and a decrease in cell inclusions, and the cell wall appeared thinner with blurred boundaries. In addition, tetramycin destroyed cell membrane integrity, resulting in the leakage of cellular components such as nucleic acids and proteins in mycelial suspensions. Moreover, tetramycin also caused cell wall lysis by enhancing the activities of chitinase and ß-1,3-glucanase and inducing the overexpression of related chitinase gene (Chit) and ß-1,3-glucanase gene (ß-1,3-glu) in A. alternata. In field trials, tetramycin not only decreased the incidence of kiwifruit rot but also create a beneficial living space for kiwifruit growth. Overall, this study indicated that the application of tetramycin could serve as an alternative measure for the management of kiwifruit rot.

Natural product osthole can significantly disrupt cell wall integrity and dynamic balance of Fusarium oxysporum.

Dendrobium officinale Kimura et Migo is a traditional Chinese herbal medicinal plant. However, the frequent occurrence of soft rot disease (SRD) is one of the most harmful diseases in D. officinale production in recent years, which can seriously affect its yield and quality. In this study, the major pathogenic fungus (SR-1) was isolated from D. officinale with typical symptoms of SRD, and was identified as Fusarium oxysporum through morphological and molecular identification. The biological activities of five natural products were determined against F. oxysporum using a mycelial growth inhibition assay. The results showed that osthole had the highest antifungal activity against F. oxysporum, with an EC50 value of 6.40 mg/L. Scanning electron microscopy (SEM) showed that osthole caused F. oxysporum mycelia to shrink and deform. Transmission electron microscopy (TEM) showed that the organelles were blurred and the cell wall was thickened in the presence of osthole. The sensitivity of F. oxysporum to calcofluor white (CFW) staining was significantly enhanced by osthole. Relative conductivity measurements and propidium iodide (PI) observation revealed that osthole had no significant effect on the cell membrane. Further experiments showed that the activity of chitinase and ß-1,3-glucanase were decreased, and expression levels of chitinase and ß-1,3-glucanase related genes were significantly down-regulated after treatment with osthole. In conclusion, osthole disrupted the cell wall integrity and dynamic balance of F. oxysporum, thereby inhibiting normal mycelial growth.

Petunia hybrida is a New Host of Pectobacterium brasiliense Associated with Soft Rot Disease in China.

Petunia hybrida is commonly cultivated for ornamental use in urban parks greening and street embellishment in China. In March 2022, 60% of P. hybrida plants cv. Wave Purple (n≈1800) from an ornamental plant nursery under natural conditions in Tianhe district (N 113°21'21", E 23°9'3.5"), Guangzhou, Guangdong Province, China, were affected with soft rot disease. The distribution of the disease was generally uniform. Infected plants initially exhibit small water-soaked lesions at the base of the stem, which then extended to the leaves. Eventually the diseased plant collapsed and died. Nine diseased plants were collected, and affected tissues cut into small fragments (5 × 5 mm), which were disinfested in 75% ethanol (30 s) and 2% sodium hypochlorite (60 s), followed by three rinses with sterile distilled water. The sterilized sections were macerated in 200 µl sterile water, and streaked on Luria-Bertani (LB) agar medium and incubated at 28°C for 48 h. Single colonies were restreaked three times to obtain purified isolation. Sixteen bacterial strains with similar morphology were isolated, and their colonies were yellowish white, round, and convex with smooth surfaces on LB agar plate. The representative strain BDQ1 was selected for further analyses and the 16S rDNA gene (GenBank Accession ON982467) were amplified using primer pair 27F/1492R, revealed above 99% sequence identity with some Pectobacterium brasiliense isolates (GenBank Accession Nos. CP046380(1421/1422), MN393966(1419/1422), and CP020350(1419/1422)) using BLASTn. A multilocus phylogenetic analysis by neighbor-joining method (1,000 bootstrap values) based on six housekeeping gene sequences of gyrA (GenBank Accession No. ON995454), icdA (ON995455), mdh (ON995456), mtlD (ON995457), proA (ON995458), and rpoS genes (ON995459) (Ma et al. 2007; Waleron et al., 2008). The results of phylogenetic analysis showed BDQ1 strain belong to the P. brasiliense clade. Pathogenicity tests were performed on ten healthy P. hybrida cv. Wave Purple plants by injecting 10 µl of bacterial suspensions of BDQ1 (108 CFU/ml) into the stems; another 10 healthy control plants were injected with 10 µl of sterile water. All plants were grown at 25-30°C and 60% humidity in natural light/dark cycle. After 3 d, all inoculated plants showed soft rot symptoms resembling to those observed in the nursery, while control plants remained healthy. Bacteria were successfully reisolated from the symptomatic tissues and identified to be P. brasiliense by PCR mentioned above. P. brasiliense is considered a very aggressive pathogen, which has been reported in Eurasia and Africa (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing bacterial soft rot on P. hybrida in China. This pathogen may pose threat to P. hybrida production in area with warmand humid climate in China. The current study expands the known host range of P. brasiliense and helped raise attention on controlling pathogen spread.

First Report of Soft Rot Caused by Pectobacterium polaris on Lettuce in China.

Lettuce (Lactuca sativa) is a leafy vegetable that belongs to the family Asteraceae. It is widely cultivated and consumed around the world. In May 2022, lettuce plants (cv. 204) showing soft rot symptoms were observed in greenhouses in Fuhai District (25°18'N, 103°6'E), Kunming City, Yunnan Province, China. The disease incidence in three greenhouses (0.3 ha in size) was between 10% to 15%. The lower parts of the outer leaves showed brown and water-soaked symptoms, but at the same time the roots were asymptomatic. Sclerotinia species can cause soft decay on lettuce leaves, known as lettuce drop, which can produce symptoms partially resembling those of bacterial soft rot (Subbarao 1998). The absence of white mycelium or black sclerotia on the leaf surfaces of diseased plants indicated that Sclerotinia species were not responsible for the disease. Instead, it is more likely that bacterial pathogens were the cause. Fourteen diseased plants were sampled from three greenhouses, and potential pathogens were isolated from the leaf tissues of six plant individuals. Leaf samples were cut into pieces ca. 0.5 cm in length. The pieces were then surface-sterilized by dipping in 75% ethanol for 60 sec, followed by three successive rinses using sterile distilled water. The tissues were immersed in 250 µl of 0.9% saline in 2 mL microcentrifuge tubes and gently pressed down with grinding pestles for 10 sec. The tubes were let stand still for 20 min. Aliquots (20 µl) 100-fold dilutions of the tissue suspensions were plated onto Luria-Bertani (LB) plates and incubated at 28°C for 24 h. Three single colonies were picked from each LB plate and restreaked five times for purity. After purification, eighteen strains were obtained, and nine of these were identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991). Six out of nine strains (6/9) belonged to the genus Pectobacterium (OP968950-OP968952, OQ568892- OQ568894), two strains (2/9) belonged to the genus Pantoea (OQ568895 and OQ568896), and one strain (1/9) belonged to Pseudomonas sp. (OQ568897). Since the Pectobacterium strains shared identical 16S rDNA sequence, strains CM22112 (OP968950), CM22113 (OP968951) and CM22132 (OP968952) were selected as representative strains for further testing. The 16S rDNA sequences of Pectobacterium strains were 100% identical to that of the P. polaris strain NIBIO 1392 (NR_159086.1). To identify the strains to the species level, multilocus sequence analysis (MLSA) was performed using sequences of six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (OP972517-OP972534) (Ma et al. 2007; Waleron et al. 2008). Phylogenetic analysis showed that the strains clustered with P. polaris type strain NIBIO1006T (Dees et al. 2017). They were all capable of utilizing citrate, which is an important biochemical feature in distinguishing P. polaris from its most closely related sister species P. parvum (Pasanen et al. 2020). Lettuce plants (cv. 204), at the rosette stage, were inoculated with the strains CM22112 and CM22132 by injecting 100 µl of bacterial suspensions (107 CFU·mL-1) into the lower parts of the leaf; for the controls, 100 µl of saline was used instead. Inoculated plants were incubated at room temperature (23°C) and 90% relative humidity. Five days after inoculation, only the bacteria-inoculated lettuce showed severe soft rot symptoms. Similar results were observed in two independent experiments. Bacterial colonies were obtained from the infected lettuce leaves, which showed identical sequences to P. polaris strains CM22112 and CM22132. Therefore, these strains fulfilled Koch's postulates for lettuce soft rot. P. polaris is prevalent on potato in many countries (Dees et al. 2017). To our knowledge, this is the first report of P. polaris causing soft rot on lettuce in China. This disease could seriously affect the appearance and saleability of lettuce. Further research on the epidemiology and management strategies of the disease is needed.

High-Quality Genome Resource of Gilbertella persicaria Causing Peach Soft Rot.

Peach soft rot caused by Gilbertella persicaria is an economically important disease. Here, we report a high-quality complete and annotated genome sequence of G. persicaria strain TFLB-J, isolated from peach fruit in Yuanyang county of Henan Province, China. The assembly consists of 91 scaffolds with an estimated genome size of 33.59 Mb and N50 length of 0.92 Mb, encoding 13,296 predicted protein-coding genes. The whole-genome sequence could provide gene resources for further study of pathogenic effectors and comparative genomics of peach soft rot pathogens.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Slawiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

First Report of Pectobacterium aroidearum Causing Soft Rot of Pinellia ternata in China.

Pinelliae rhizoma is the dried tuber of Pinellia ternata (Thunb.) Breit., and has been used for thousand of years in traditional Chinese medicine as an antivomit, anticough, and analgesic (Ying et al. 2007). In September 2022, P. ternata planted in Bijie, Guizhou Province, showed severe soft rot symptoms with incidence of about 50%. The diseased plants showed water-soaked symptoms and produced a foul soft rot smell, and finally the whole plant collapsed. Lesions were first observed at the tip of a leaf or wound, and symptoms of the disease spread rapidly, with the entire plant collapsing and dying within a week. The tissue sections of six plants with typical symptoms from the diseased field were disinfected with 75% ethanol for 30 seconds and 0.3% NaClO for 3 minutes. The tissue sections were then washed with sterile water for three times. A small piece of tissue (5x5mm) was removed from the edge of the lesion and mashed in a 1.5 ml centrifuge tube containing 20 µl of sterile water. The tissue liquid was then diluted 100 times with prepared sterile water. The bacteria were streaked on LB (tryptone/yeast extract/NaCl) AGAR medium and cultured at 37°C for 48 h (Kravitz, 1962). Isolated colonies were streaked on Luria-Bertani (LB) AGAR medium to obtain single colonies for further identification. A total of 13 representative isolates were selected for PCR amplification using primers targeting the conserved region of the 16S rDNA gene, which were in turn analyzed via the BLASTn search engine on the NCBI website. The results of the analysis revealed that seven of the isolates were similar to P. aroidearum strain SCRI 109 (GenBank accession no. NR_159926), with strain BX13 exhibiting the highest similarity to P. aroidearum (99.93% similarity), and therefore, this strain was selected for further investigation. The strain BX13 was incubated on LB solid medium for 24 h at 37 °C, and the single colonies were creamy white, translucent and round, slightly elevated in the center, with smooth surfaces and neat edges (Figure S1 B1). Then,the Scanning Electron Microscope revealed that the thalli of strain BX13 were short rod-shaped and somewhat blunt round at both ends (Figure S1 B2). The steward genes (icdA, gapA, proA) of BX13 were amplified and sequenced for further identification. The sequences of the amplified fragments were all deposited in GenBank 16S rDNA (OQ874505,) icdA (OQ954122)，gapA (OQ954123), proA (OQ954124). Sequence analysis using the BLASTn program at the NCBI revealed gene icdA, gapA, and proA had 100% identity to P. aroidearum strain QJ002 (GenBank accession no. CP090597).. Meanwhile, a maximum likelihood phylogenetic tree was constructed based on multigene sequence analysis of BX13 16S rDNA and steward genes (gapA, icdA, proA) by MEGA X (Liang et al. 2022). Phylogenetic results also showed that BX13 and P. aroidearum strain QJ002 gathered in the same clade(Figure S2). Accordingly, the morphological and molecular characteristics of strain BX13 indicate that it is P. aroidearum. (Nabhan S., et al.2013,Xu et al. 2020). In order to confirm the pathogenicity of strain BX13, a bacterial suspension containing 107 CFU/ml (10 ml/ inoculation point) was injected into the base of a one-week-old P. ternata stems, control seedlings were inoculated with sterile water, inoculated and control seedlings (each of six plants) were kept in a growth chamber maintained at 26°C with a relative humidity range of 70% to 80%. Plants were watered as needed. After 3 days, the stem base of the plants inoculated with bacteria solution showed water-soaked necrosis and stems began to rot, while the plants inoculated with water did not show this symptom. The strains were then successfully re-isolated from the symptomatic P. ternata. Then the strain re-isolated was identified using the BLASTn program at the NCBI and found that it has the same 16S rDNA, icdA, gapA, and proA sequences as strain BX13, thus completing the Koch's postulates. To our knowledge, this is the first report of P. aroidearum causing P. ternata soft rot in China, which expands its known host range. Accordingly, this study provides essential information for the breeding of P. ternata resistant to bacterial soft rot and the development of control measures in China.

First Report of Cucurbita Blossom Blight and Fruit Rot Caused by Choanephora cucurbitarum in Mexico.

In December 2022, blossom blight, abortion, and soft rot of fruits were observed on Cucurbita pepo L. var. Zucchini in Mexico under greenhouse conditions (temperatures of 10 to 32°C and relative humidity up to 90%). The disease incidence in about 50 plants analyzed was around 70% with a severity of nearly 90%. Mycelial growth on flower petals and fruit rot with brown sporangiophores was observed. Ten disinfested fruit tissues in 1% NaClO for 5 min and then rinsed twice in distilled water from the lesion edges were placed on potato dextrose agar culture medium (PDA) supplemented with acid lactic and then, the morphological characterization was carried out in V8 agar medium. After 48 h of growth at 27°C, the colonies were pale yellow with diffuse cottony mycelia that were non-septate and hyaline and produced both sporangiophores bearing sporangiola and sporangia. The sporangiola were brown, ranged from ellipsoid to ovoid, and had longitudinal striations that measured 22.7 to 40.5 (29.8) µm x 16.08 to 21.9 (14.5) µm long and wide, respectively (n=100). The sporangia were subglobose, had a diameter of 127.2 to 281.09 (201.7) µm (n=50), and contained ovoid sporangiospores that measured 26.5 to 63.1 (46.7) µm x 20.07 to 34.7 (26.3) µm long and wide, respectively (n=100) which had hyaline appendages at the ends. Based on these characteristics, the fungus was identified as Choanephora cucurbitarum (Ji-Hyun et al. 2016). For molecular identification, DNA fragments for the internal transcribed spacer (ITS) and the large subunit rRNA 28S (LSU) regions for two representative strains (CCCFMx01 and CCCFMx02) were amplified and sequenced with the primer pairs ITS1-ITS4 and NL1-LR3 (White et al. 1990; Vilgalys and Hester 1990). The ITS and LSU sequences were deposited in GenBank database (Accession numbers OQ269823-24 and OQ269827-28, respectively) for both strains. The Blast alignment showed from 99.84 to 100% identity with Choanephora cucurbitarum strains JPC1 (MH041502, MH041504), CCUB1293 (MN897836), PLR2 (OL790293), and CBS 178.76 (JN206235, MT523842). To confirm the specie identification, the evolutionary analyses were conducted from the concatenated sequences of the ITS and LSU of C. cucurbitarum and other mucoralean species with the Maximum Likelihood method and Tamura-Nei model included in the software MEGA11. The pathogenicity test was demonstrated using five surface-sterilized zucchini fruits inoculated with a sporangiospores suspension containing a concentration of 1 x 105 esp/mL on two sites per fruit (20 µL each) that previously were wounded with a sterile needle. For fruit control, 20 µL of sterile water was used. Three days after inoculation under humidity conditions at 27°C, white mycelia and sporangiola growth with a soaked lesion were observed. That fruit damage was not observed on the control fruits. C.cucurbitarum was reisolated from lesions on PDA and V8 medium which was confirmed by morphological characterization fulfilling Koch's postulates. Blossom blight, abortion, and soft rot of fruits caused by C. cucurbitarum were observed on Cucurbita pepo and C. moschata in Slovenia and Sri Lanka (Zerjav and Schroers 2019; Emmanuel et al. 2021). This pathogen has the capability to infect a wide variety of plants worldwide (Kumar et al. 2022; Ryu et al. 2022). There are no reports of C. cucurbitarum causing agricultural losses in Mexico, and this is the first report causing the disease symptoms in Cucurbita pepo in this country; however, this fungus was found in the soil of papaya-producing areas and it is considered an important plant pathogenic fungus. Therefore, strategies for their control are highly recommended to avoid spreading the disease (Cruz-Lachica et al. 2018).