Metabolomic Fingerprinting of Potato Cultivars Differing in Susceptibility to Spongospora subterranea f. sp. subterranea Root Infection.

Plants defend themselves from pathogens by producing bioactive defense chemicals. The biochemical mechanisms relating to quantitative resistance of potato to root infection by Spongospora subterranea f. sp. subterranea (Sss) are, however, not understood, and are not efficiently utilized in potato breeding programs. Untargeted metabolomics using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to elucidate the biochemical mechanisms of susceptibility to Sss root infection. Potato roots and root exudate metabolic profiles of five tolerant cultivars were compared with those of five susceptible cultivars, following Sss inoculation, to identify tolerance-related metabolites. Comparison of the relative metabolite abundance of tolerant versus susceptible cultivars revealed contrasting responses to Sss infection. Metabolites belonging to amino acids, organic acids, fatty acids, phenolics, and sugars, as well as well-known cell wall thickening compounds were putatively identified and were especially abundant in the tolerant cultivars relative to the susceptible cultivars. Metabolites known to activate plant secondary defense metabolism were significantly increased in the tolerant cultivars compared to susceptible cultivars following Sss inoculation. Root-exuded compounds belonging to the chemical class of phenolics were also found in abundance in the tolerant cultivars compared to susceptible cultivars. This study illustrated that Sss infection of potato roots leads to differential expression of metabolites in tolerant and susceptible potato cultivars.

Population Biology and Genetic Variation of Spongospora subterranea f. sp. subterranea, the Causal Pathogen of Powdery Scab and Root Galls on Potatoes in South Africa.

Spongospora subterranea f. sp. subterranea, causal agent of powdery scab and root galls of potatoes, occurs worldwide and is responsible for quality and yield losses in potato production in South Africa. Despite being one of the most important potato pathogens in South Africa, little information is available on the genetic structure and diversity of S. subterranea f. sp. subterranea, which could provide insight into the factors shaping its evolution and the role of inoculum sources in disease development. A total of 172 samples were collected from four potato growing regions in South Africa. An additional 27 samples obtained from Colombia were included for comparative purposes. The samples were screened against six informative microsatellite (simple-sequence repeat) markers. Of the 172 samples obtained from potato growing regions in South Africa, there were 75 multilocus genotypes (MLGs), only 16 of which were shared between potato growing regions, indicating substantial gene flow and countrywide dispersal of the pathogen. The presence of common MLGs among the root- and tuber-derived samples indicated a lack of specialization of S. subterranea f. sp. subterranea to either tuber or root infection. Nei's unbiased estimates of gene diversity for the clone-corrected data were low and ranged from 0.24 to 0.38. Analysis of molecular variance and discriminant analysis of principal components showed no population differentiation between different potato growing regions in South Africa and between root- and tuber-derived genotypes. The presence of MLGs, high considerable genotypic diversity, and failure to reject the null hypothesis of random mating in most populations are indicative of some kind of recombination, either sexual or asexual, in these S. subterranea f. sp. subterranea populations. Information from this study provides new insights into the genetic structure and diversity of S. subterranea f. sp. subterranea in South Africa. Continuous monitoring of the pathogen population dynamics will be helpful in implementing effective region-specific management strategies for the pathogen, especially in the development of resistant potato cultivars.

Relative Contribution of Seed Tuber- and Soilborne Inoculum to Potato Disease Development and Changes in the Population Genetic Structure of Rhizoctonia solani AG 3-PT under Field Conditions in South Africa.

Understanding the contribution of seed tuber- and soilborne inocula of Rhizoctonia solani AG 3-PT in causing potato disease epidemics is an important step in implementing effective management strategies for the pathogen. A 2-year study was conducted to evaluate the contribution of each source of inoculum using an integrative experimental approach combining field trials and molecular techniques. Two distinct sets of genetically marked isolates were used as seed tuberborne and soilborne inocula in a mark-release-recapture experiment. Disease assessments were done during tuber initiation and at tuber harvest. Both inoculum sources were found to be equally important in causing black scurf disease, whereas soilborne inocula appeared to be more important for root and stolon infection, and seedborne inocula contributed more to stem canker. However, seed tuber-transmitted genotypes accounted for 60% of the total recovered isolates when genotyped using three polymerase chain reaction restriction fragment length polymorphism markers. The changes in population structure of the experimental R. solani population over the course of the growing season and across two growing seasons were investigated using eight microsatellite markers. The populations at different sampling times were somewhat genetically differentiated, as indicated by Nei's gene diversity (0.24 to 0.27) and the fixation index (FST). The proportion of isolates with genotypes that differed from the inoculants ranged from 13 to 16% in 2013 and 2014, respectively, suggesting the possibility of emergence of new genotypes in the field. Because both soilborne and tuberborne inocula are critical, it is important to ensure the use of pathogen-free seed tubers to eliminate seed tuberborne inoculum and the introduction of new genotypes of R. solani for sustainable potato production in South Africa.

Ralstonia solanacearum needs Flp pili for virulence on potato.

Type IV pili are virulence factors in various bacteria. Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Although type IVa pili have been implicated in the virulence of Ralstonia solanacearum, type IVb pili have not previously been described in this plant pathogen. Here, we report the characterization of two distinct tad loci in the R. solanacearum genome. The tad genes encode functions necessary for biogenesis of the Flp subfamily of type IVb pili initially described for the periodontal pathogen Aggregatibacter actinomycetemcomitans. To determine the role of the tad loci in R. solanacearum virulence, we mutated the tadA2 gene located in the megaplasmid that encodes a predicted NTPase previously reported to function as the energizer for Flp pilus biogenesis. Characterization of the tadA2 mutant revealed that it was not growth impaired in vitro or in planta, produced wild-type levels of exopolysaccharide galactosamine, and exhibited swimming and twitching motility comparable with the wild-type strain. However, the tadA2 mutant was impaired in its ability to cause wilting of potato plants. This is the first report where type IVb pili in a phytopathogenic bacterium contribute significantly to plant pathogenesis.

Role of Polyphenol Oxidase, Peroxidase, Phenylalanine Ammonia Lyase, Chlorogenic Acid, and Total Soluble Phenols in Resistance of Potatoes to Soft Rot.

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya spp. cause soft rot of potato (Solanum tuberosum) worldwide. Plants respond to bacterial invasion by activating defense responses associated with accumulation of several enzymes and inhibitors, which prevent pathogen infection. This study focused on the role of polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), chlorogenic acid, and total soluble phenols in imparting resistance to soft rot pathogens. Seven and 11 varieties grown by farmers in South Africa and Zimbabwe, respectively, were used in the study. The results showed significantly higher (P < 0.001) enzyme activity of PPO and PAL as well as higher concentrations of chlorogenic acid and total soluble phenols in Vanderplank, Pentland Dell, M69/11, Romano, M59/20, and MondialZw. PAL activity increased significantly with time in all varieties, and the highest activity was recorded 8 h after cutting. The resistance of the varieties was correlated with high PPO and PAL enzyme activity as well as increased concentrations of chlorogenic acid and total soluble phenols. PPO, POD, and PAL activities increased significantly in wounded and inoculated tubers. These findings show that PAL, PPO, POD, chlorogenic acid, and total soluble phenols play a role in imparting resistance to potato soft rot infection.

Plant-soil feedback responses of four dryland crop species under greenhouse conditions.

Plant-soil feedbacks (PSFs) give a mechanistic understanding on how soil properties established by previous plant species go on to influence the performance of the same or different species in monoculture, intercropping or crop rotation systems. We hypothesized that different dryland crops such as Zea mays L., Helianthus annuus L., Phaseolus vulgaris L., and Glycine max L. (Merr.) will have soil legacies that are related to the crop type. We used a two-phase experiment to test plant performance in soils previously cultivated with the same or different plant species under greenhouse conditions. The positive plant growth for all species in their own soil microbiota suggests that mutualists had a greater impact on plant performance than pathogens. The consistent positive soil-feedback results of P. vulgaris were strongly associated with their own beneficial soil microbiota, meaning that the conditioning phase legacy of mutualists and decomposers were more significant than pathogens under monoculture. Despite successful nodulation in sterilized and inoculated soils, G. max unexpectedly showed neutral and insignificant positive plant feedbacks, respectively. Helianthus annuus was superior to other crop species in creating active carbon stocks and an enzymatically active soil for the next crop. Microbial biomass results suggest that raising fungal relative to bacterial biomass can be achieved by increasing the frequency of H. annuus in rotation sequences. However, more studies are necessary to evaluate whether these elevated ratios promote or depress plant performance under field conditions. This study showed that relative to other dryland crops, H. annuus seems to have the potential of increasing fungal to bacterial ratios, raising legacies in active carbon stocks and soil microbial activity that may be crucial to successional planting in dryland systems.

Temporal shifts of fungal communities in the rhizosphere and on tubers in potato fields.

Soil fungal communities perform important ecological roles determining, at least in part, agricultural productivity. This study aimed at examining the fungal community dynamics in the potato rhizosphere across different development stages in two consecutive growing seasons (winter and summer). Microbial fingerprinting of rhizosphere soil samples collected at pre-planting, tuber initiation, flowering and at senescence was performed using ARISA in conjunction with Next Generation Sequencing (Illumina MiSeq). The epiphytic fungal communities on tubers at harvest were also investigated. Alpha-diversity was stable over time within and across the two seasons. In contrast, rhizospheric fungal community structure and composition were different between the two seasons and in the different plant growth stages within a given season, indicating the significance of the rhizosphere in shaping microbial communities. The phylum Ascomycota was dominant in the potato fungal rhizosphere, with Operational Taxonomic Units (OTUs) belonging to the genus Peyronellaea being the most abundant in all samples. Important fungal pathogens of potato, together with potential biological control agents and saprophytic species, were identified as indicator OTUs at different plant growth stages. These findings indicate that potato rhizosphere fungal communities are functionally diverse, which may contribute to soil health.

Population genetic structure of Rhizoctonia solani AG 3-PT from potatoes in South Africa.

Rhizoctonia solani AG 3-PT is an important potato pathogen causing significant yield and quality losses in potato production. However, little is known about the levels of genetic diversity and structure of this pathogen in South Africa. A total of 114 R. solani AG 3-PT isolates collected from four geographic regions were analysed for genetic diversity and structure using eight microsatellite loci. Microsatellite analysis found high intra-population genetic diversity, population differentiation and evidence of recombination. A total of 78 multilocus genotypes were identified with few shared among populations. Low levels of clonality (13-39 %) and high levels of population differentiation were observed among populations. Most of the loci were in Hardy-Weinberg equilibrium and all four populations showed evidence of a mixed reproductive mode of both clonality and recombination. The PCoA clustering method revealed genetically distinct geographic populations of R. solani AG 3-PT in South Africa. This study showed that populations of R. solani AG 3-PT in South Africa are genetically differentiated and disease management strategies should be applied accordingly. This is the first study of the population genetics of R. solani AG 3-PT in South Africa and results may help to develop knowledge-based disease management strategies.

Genetic Diversity Among Alternaria solani Isolates from Potatoes in South Africa.

Genetic diversity among isolates of Alternaria solani, the causal agent of early blight of potato, from various potato-growing regions in South Africa (SA), was determined using virulence assays, vegetative compatibility (VC) tests, and random amplified microsatellite (RAMS) primers. The virulence assays showed low virulence levels for the largest part of the population, but failed to otherwise characterize the population diversity. The VC tests revealed 19 VC groups (VCGs), indicating a relatively high level of diversity among the isolates. There was little correlation between geographic origin of isolates and VCGs. Analysis of RAMS profiles revealed 27% genetic diversity among 46 isolates. This value is relatively high for an asexually reproducing fungus, but is similar to values obtained previously by authors studying A. solani. Distance analysis of the RAMS profiles also provided no evidence for geographical clustering of isolates. VCG and RAMS profiles indicated that isolates are randomly spread across SA. This fact, together with the high diversity of A. solani in SA, indicates that the fungus has a high potential to adapt to resistant cultivars or fungicides. This information can aid in the breeding and deployment of A. solani-resistant potato varieties, and in early blight disease management in SA.

The role of secretion systems and small molecules in soft-rot Enterobacteriaceae pathogenicity.

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.