Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei.

Bacteriophages have emerged as promising alternatives to pesticides for controlling bacterial pathogens in crops. Among these pathogens, Streptomyces stelliscabiei (syn. S. stelliscabiei) is a primary causative agent of potato common scab (PCS), resulting in substantial global economic losses. The traditional management methods for PCS face numerous challenges, highlighting the need for effective and environmentally friendly control strategies. In this study, we successfully isolated three novel bacteriophages, namely Psst1, Psst2, and Psst4, which exhibited a broad host range encompassing seven S. stelliscabiei strains. Morphological analysis revealed their distinct features, including an icosahedral head and a non-contractile tail. These phages demonstrated stability across a broad range of temperatures (20-50°C), pH (pH 3-11), and UV exposure time (80 min). Genome sequencing revealed double-stranded DNA phage with open reading frames encoding genes for phage structure, DNA packaging and replication, host lysis and other essential functions. These phages lacked genes for antibiotic resistance, virulence, and toxicity. Average nucleotide identity, phylogenetic, and comparative genomic analyses classified the three phages as members of the Rimavirus genus, with Psst1 and Psst2 representing novel species. All three phages efficiently lysed S. stelliscabiei in the liquid medium and alleviated scab symptom development and reduced pathogen abundance on potato slices. Furthermore, phage treatments of radish seedlings alleviated the growth inhibition caused by S. stelliscabiei with no disease symptoms. In soil potted experiments, phages significantly reduced disease incidence by 40%. This decrease is attributed to a reduction in pathogen density and the selection of S. stelliscabiei strains with reduced virulence and slower growth rates in natural environments. Our study is the first to report the isolation of three novel phages that infect S. stelliscabiei as a host bacterium. These phages exhibit a broad host range, and demonstrate stability under a variety of environmental conditions. Additionally, they demonstrate biocontrol efficacy against bacterial infections in potato slices, radish seedlings, and potted experiments, underscoring their significant potential as biocontrol agents for the effective management of PCS.

Species diversity of Pectobacterium spp. causing potato aerial stem rot in China.

Pectobacterium spp. are the primary causative agents of aerial stem rot in potatoes in China. A nationwide survey revealed the widespread occurrence of aerial stem rot in the northern, southern, and southwestern cultivation regions, with occurrence rates ranging from 1% to 60%. In total, 36 strains were isolated and identified at the species level using multi-locus sequence analysis of six housekeeping genes (rpoS, proA, gapA, icdA, gyrA, and mdh). Genome sequencing was conducted on one representative strain for each species, and further confirmation of their identities was achieved through ANI and isDDH analysis. Five Pectobacterium species were identified, namely Pectobacterium atrosepticum, Pectobacterium brasiliense, Pectobacterium carotovorum, Pectobacterium polaris and Pectobacterium punjabense, with P. atrosepticum and P. brasiliense being the most widely distributed. Pathogenicity tests demonstrated that, among the strains isolated in this study and those obtained from other studies, P. atrosepticum and P. brasiliense are also the most virulent species. To the best of our knowledge, this is the first nationwide study describing the diversity and distribution of Pectobacterium spp. affecting potatoes in China. The information gathered will be utilized for disease diagnosis and the development of pathogen-specific integrated pest management (IPM) strategies to protect potato production.

First Report of Pectobacterium parvum Causing Diseases on Potato in Inner Mongolia, China.

Soft rot enterobacterial plant pathogens Pectobacterium spp. and Dickeya spp. have caused devastating blackleg, aerial stem rot, and soft rot of potato tubers (Charkowski 2018). In August 2021, potato plants (cv. Xisen6# or Maiken1) with blackleg or aerial stem rot symptoms were observed in two commercial fields in Xinghe County, Ulanqab City, Inner Mongolia Autonomous Region, China. The plants were wilted, and the crown stem showed gradual degradation and browning. The disease incidence was around 3 to 7% and 20 to 25% in Xinghe Zhangyou Village (33 ha) and Bianjia Village (7 ha), respectively. Pathogens were isolated on crystal violet pectate agar (CVP) plates (Ge et al. 2018). Briefly, symptomatic stem tissues were surface sterilized in 75% ethanol, ground, then serial dilutions were cultured on CVP plates (Handique et al. 2022). The plates were incubated at 28oC for 2 days. Pure colonies of Pectobacterium spp. were obtained from the pits of CVP plates and sequenced for identification using the universal 16S rRNA gene primers 27F/1492R (Monciardini et al. 2002). Results of the comparison of 16S sequences against NCBI GenBank showed 100% sequence identity to P. parvum FN20211T (CP087392.1) type strain for the three colonies designated as ZRIMU1006 (1542/1542 bp), ZRIMU1019 (1542/1542 bp), and ZRIMU1020 (1542/1542 bp). Sequences were deposited under accession numbers OP941529, OP941525, OP941526, respectively. Additionally, six housekeeping gene sequences were used to confirm identification at the species level and were uploaded to GenBank: fusA (OP793177, OP793171, OP793172), gapA (OP793221, OP793216, OP793217), gyrB (OP793265, OP793259, OP793260), infB (OP793310, OP793304, OP793305), pgi (OP793355, OP793349, OP793350), and rplB (OP793400, OP793394, OP793395). Phylogenetic trees constructed using the MEGA X program of concatenated sequences of the housekeeping genes sequences show that the three isolates grouped with P. parvam FN20211T, confirming that they are P. parvam. Pathogenicity tests for stem rot were done by injecting a bacterial suspension into potato seedlings (cv. Favorita) grown from seed tubers. The tubers were planted in perlite potting mix and 3 weeks after emergence, a 100 µl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution was injected at the stem base. The bacterial injection experiment was performed twice in a greenhouse with five plants inoculated per bacterial strain. Plants were covered with plastic bags to maintain 100% humidity at 25°C for 2 days. Seven days after inoculation, the inoculated area of the seedlings had rotted or turned black, while the controls remained symptomless. Symptomatic tissues from each strain were processed as above and placed on CVP plates to reisolate the Pectobacterium spp. 16S rRNA sequence analysis confirmed the bacteria to be similar in sequence to the inoculated strains, thus completing Koch's postulates. Soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml) on tuber slices. The infected tubers rotted, while the controls were symptomless. The vacuum infiltration method on tuber that is used to test pathogens for blackleg did not result in the development of blackleg symptoms. Briefly, five tubers were inoculated with the pathogen by vacuum infiltration and planted in potting mix. The plants showed wilting after emergence, but no blackleg symptoms were observed. Recently, multiple new Pectobacterium species including P. parmentieri, P. polaris, and P. punjabense, were identified to cause potato disease in different provinces of China (Cao et al. 2021; Handique et al. 2022). In China, P. parvum was first isolated from Brassica, and in the year 2022, it was reported to cause aerial stem-rot on potato in Hebei province in China (Wang et al. 2022). Inner Mongolia is a major potato-seed-producing province and the incidence of new strains of Pectobacterium in the province causing aerial stem rot and soft rot of tuber might cause a reduction in seed production. This report will draw attention to the management of P. parvum by the seed-producing companies in Inner Mongolia which distributes the seed throughout China.

First Report of Streptomyces niveiscabiei Causing Potato Common Scab in Shanxi and Gansu, China.

Multiple species of Streptomyces cause common scab disease in potato (Solanum tuberosum) (Kämpfer et al. 1991). Potato tubers (cv. Jinshu1 #5 and Longshu #6) with severe pitted common scab symptoms were observed from two farms in Chaozhou in Shanxi Province and in Tianzhu in Gansu Province during the national disease survey of bacterial diseases of potatoes in 2021. The disease incidence was around 30% on the 6.7 ha of the Chaozhou farm and 10% on the 0.7 ha on the Tianzhu farm. Three tubers with scab symptoms were surface disinfested with 3% sodium hypochlorite for 1 min. The symptomatic tissue was then ground in sterile water. Serially diluted ground samples were cultured on Streptomyces ISP Medium 5 agar plates (Shirling and Gottlieb 1966) and incubated at 280C for 5 days. Eight pure Streptomyces colonies were obtained and sequenced for identification using the universal 16S rRNA gene primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (Monciardini et al. 2002) by colony PCR. Blast results of the sequences against the NCBI GenBank for the eight isolates, ZRIMU1508, ZRIMU1510, ZRIMU1511, ZRIMU1512, ZRIMU1514, ZRIMU1515, ZRIMU1516 and ZRIMU1530 (Accession numbers: OP941573 - OP941580), showed more than 99% sequence identity to S. niveiscabiei NRRLB-24457T type strain. Additionally, 12 housekeeping gene sequences, acnA (OP997624 - OP997625), atpD (OP997622 - OP997623), dnaN (OP997620 - OP997621), gap (OP997618 - OP997619), gyrA (OP997614 - OP997615), gyrB (OP997612 - OP997613), infB (OP997610- OP997611), mdh (OP997608 - OP997609), recA (OP997602 - OP997603), rplB (OP997600 - OP997601), rpoB (OP997598- OP997599), and trpB (OP997594 - OP997595), were extracted from the genome sequences of two strains, ZRIMU1510 and ZRIMU1530, and uploaded to GenBank. Genes for pathogenicity, txtA (OP997593 - OP997594), tomA (OP997596 - OP997597) and Nec1(OP997606 - OP997607), were also identified from the genome sequence and uploaded to GenBank. The housekeeping genes and the pathogenicity genes showed over 98% identity with S. niveiscabiei. Phylogenetic trees were constructed using concatenated housekeeping gene sequences (Kumar et al. 1994) and the cladogram showed that the isolates ZRIMU1510 and ZRIMU1530 grouped with the type strain NRRLB-24457T. Pathogenicity tests were done by drench application of 100 ml spore suspensions (104 CFU/ml) of ZRIMU1530, ZRIMU1510, or phosphate buffer into pots with potato plants (cv. Favorita) grown in potting mix. Five tubers were planted and inoculated with each pathogen or phosphate buffer as the negative control. The plants were then placed in a greenhouse with 12 h of light per day, irrigated regularly, and harvested after 3 months. The newly formed tubers were checked for disease symptoms. Tubers from pots inoculated with ZRIMU1530 and ZRIMU1510 exhibited typical symptoms of common scab with raised corky lesions with deep pits, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed to be identical to the original isolates by 16S rRNA gene sequences, thus completing Koch's postulates. The pot experiment was conducted twice: first in May 2022 and second in February 2023. To our knowledge, this is the first report of S. niveiscabiei causing common scab of potato in Shanxi and Gansu, China. S. niveiscabiei was first reported in Korea (Park et al. 2003) and this report will draw attention to the study and management of scab pathogens in China.

First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 µl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.

First Report of Streptomyces stelliscabiei Causing Potato Common Scab in Guizhou Province, China.

Potato (Solanum tuberosum L.) common scab can be caused by multiple pathogenic Streptomyces spp. worldwide. Potato tubers (cv. Favorita) with severe pitted common scab symptoms were observed at a small farm (2 hectares) during harvest in Anshun, Guizhou province in early May 2020. The disease incidence was around 10%, and symptomatic samples were collected to isolate the pathogen. Two isolates, ZR-IMU141 and ZR-IMU146 (Accession number MW995958 and MW995959 respectively), showed more than 99% sequence identity to S. stelliscabiei sequences (Accession No. HM018085). Five house-keeping genes for multi-locus sequence analyze (MLSA) of Streptomycetaceae were amplified, sequenced and uploaded to NCBI: atpD (MZ343164 and MZ343165), gyrB (MZ343162 and MZ343163), recA (MZ343166 and MZ343167), rpoB (MZ343168and MZ343169) and trpB (MZ343170 and MZ343171). All the genes show over 98% identity with S. stelliscabiei. Phylogenetic trees of 16S rRNA gene sequence and multi-locus sequence analysis (MLSA) were constructed. The two isolates contain pathogenicity genes txtAB, nec1, and tomA, which was confirmed by PCR. To complete Koch's postulates, 9 potato seedlings (cv. Favorita, 15 centimeters high), were transferred to new pots and inoculated with spore suspensions of ZR-IMU141 and ZR-IMU146 (104 CFU/ml), or water as a negative control. Two months later, potato tubers inoculated with either ZR-IMU141 or ZR-IMU146 exhibited typical symptoms of potato common scab, such as superficial or deep, raised, pitted, or polygonal lesions like the field symptoms, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed identical to the original isolate by 16s rRNA gene sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Guizhou province, China. We believe that this report will draw attention to the study and management of the increased pool of scab pathogens in China.

First Report of Pectobacterium parmentieri Causing Blackleg on Potato in Inner Mongolia, China.

Blackleg on potato plants (Solanum tuberosum) is caused by Pectobacterium spp. and Dickeya spp. (Charkowski, 2018) worldwide. From June to August in both 2018 and 2019, cases of blackleg were investigated in potato-producing areas in Hulunbuir, Ulanqab, and Hohhot in Inner Mongolia, China. The total surveyed field area was about 200 hectares. The plants showed typical blackleg symptoms, such as black and stunted stems or curled leaves (Fig. S1), and the number of infected plants were counted. The disease showed an incidence of around 8%. Five diseased plants were collected from a 10 ha potato field with approximately 75,000 potato plants (cv. mainly Favorita and Xisen) per hectare. Two-centimeter-long samples of symptomatic stems were removed from the selected plants using a sterile scalpel. The surfaces of the samples were disinfected with 75% ethanol for 2 min. They were then rinsed with sterile distilled water and soaked in 5 ml sterile distilled water for 30 min. Aliquots of three tenfold dilutions of this solution were plated onto the crystal violet pectate agar (CVP) plate and incubated for 3 days at 28°C (Ge et al., 2018). A single bacterial colony that showed pitting on CVP plates (Fig. S2) was picked with a toothpick, streaked onto nutritional agar (She et al., 2013) to obtain pure colonies. Amplification of a 1.4-kb segment containing 16S rRNA gene was performed on the pure colonies using the universal primer set 27F/1492R (Monciardini et al., 2002). The amplicons were sequenced and submitted to the GenBank Nucleotide Basic Local Alignment Search Tool analysis. The 16S rRNA gene sequences of four isolates (GenBank accession numbers: MN626412, MN626449, MN625916, and MT235556) showed more than 99% sequence identity to Pectobacterium parmentieri type strain RNS 08-42-1A (NR_153752.1) (Fig. S3). Six housekeeping genes proA (MT427753-MT427756), gyrA (MT427757-MT427760), icdA (MT427761-MT427764), mdh (MT427765-MT427768), gapA (MT427769-MT427772), and rpoS (MT427773-MT427776) of these four isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All sequences showed 99% to 100% sequence identity with Pectobacterium parmentieri strains. Phylogenetic trees (Fig. S4) were constructed by multi-locus sequence analysis (MLSA) using MEGA 6.0 software (Tamura et al., 2013). The samples were tested against Koch's postulates on potato seedlings (cv. Favorita) by injecting 100 µl bacterial suspension (107 CFU/ml) or sterile phosphate buffered solution into the stems 2 cm above the base (Ge et al., 2018). The seedlings were incubated at 21°C and 80% humidity (She et al., 2013). Three to 5 days after inoculation, only infected seedlings showed similar symptoms as those observed in the field: the infected area turned black and rotten (Fig. S5). Bacterial colonies isolated from these symptomatic seedlings were identified using the same methods described above and were identified as inoculated Pectobacterium parmentieri strains. Blackleg on potato plants has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. brasiliense in China (Zhao et al., 2018). To our knowledge, this is the first report of blackleg of potato caused by Pectobacterium parmentieri in Inner Mongolia, China. We believe that this report will draw attention to the identification of this pathogen, which is essential to disease management.

Phytophthora nicotianae Infection of Citrus Leaves and Host Defense Activation Compared to Root Infection.

Currently, little is known about the host pathogen interaction between Phytophthora spp. and citrus roots versus leaves. Therefore, we compared the molecular events occurring in citrus roots and leaves after zoospore inoculation with Phytophthora nicotianae. We analyzed the physical characteristics and genetic responses to P. nicotianae infection of leaves and roots for susceptible and tolerant citrus rootstocks to examine the potential for leaves to model root responses to P. nicotianae infection. Leaves responded faster and stronger to P. nicotianae infection than roots, and leaves showed greater differential response than roots. In addition to differences in hormonal responses, sugar, phospholipase D (PLD), and phospholipase A (PLA) involvement in the interaction between citrus and P. nicotianae was identified. This work, for the first time, creates a solid P. nicotianae zoospore infection protocol, reports P. nicotianae infection on citrus leaves through stomata, and provides evidence that different host organs respond to the pathogen differentially in timing and magnitude. This work identifies the hormones, sugars, pathogenesis-related genes, PLDs, and PLAs that are involved in the molecular events occurring in citrus under infection of P. nicotianae zoospore, and advances our understanding of the mechanisms underlying the interaction.

Characterization of Antimicrobial-Producing Beneficial Bacteria Isolated from Huanglongbing Escape Citrus Trees.

The microbiome associated with crop plants has a strong impact on their health and productivity. Candidatus Liberibacter asiaticus (Las), the bacterial pathogen responsible for Huanglongbing (HLB) disease, lives inside the phloem of citrus plants including the root system. It has been suggested that Las negatively affects citrus microbiome. On the other hand, members of citrus microbiome also influence the interaction between Las and citrus. Here, we report the isolation and characterization of multiple putative beneficial bacteria from healthy citrus rhizosphere. Firstly, six bacterial strains showing antibacterial activity against two bacteria closely related to Las: Agrobacterium tumefaciens and Sinorhizobium meliloti were selected. Among them, Burkholderia metallica strain A53 and Burkholderia territorii strain A63 are within the ß-proteobacteria class, whereas Pseudomonas granadensis strain 100 and Pseudomonas geniculata strain 95 are within the γ-proteobacteria class. Additionally, two gram-positive bacteria Rhodococcus jialingiae strain 108 and Bacillus pumilus strain 104 were also identified. Secondly, antimicrobial activity against three fungal pathogens: Alternaria alternata, Colletotrichum acutatum, Phyllosticta citricarpa, and two oomycetes: Phytophthora nicotianae and Phytophthora palmivora. Four bacterial strains Burkholderia territorii A63, Burkholderia metallica A53, Pseudomonas geniculata 95, and Bacillus pumilus 104 were shown to have antagonistic activity against the citrus root pathogen Phytophthora nicotianae based on dual culture antagonist assays and compartmentalized petri dish assays. The four selected bacteria were sequenced. Genes involved in phosphate solubilization, siderophore production and iron acquisition, volatile organic compound production, osmoprotection and osmotic tolerance, phytohormone production, antagonism, and nutrient competition were predicted and discussed related to the beneficial traits.