First report of Ralstonia pseudosolanacearum in the Lao PDR
Ralstonia pseudosolanacearum is reported for the first time from the Lao PDR. Typical symptoms of wilt and vascular exudate were observed in potatoes and tomatoes in Paksong district, Champasak province. Pathogenicity was proven and identification was based on symptoms, commercial diagnostic test kits and sequence of the egl gene.
KeywordsBacterial wilt Potato Pseudomonas solanacearum Ralstonia solanacearum
Ralstonia solanacearum (sensu lato) is a widely distributed bacterial pathogen in tropical, sub-tropical and temperate countries causing vascular necrosis and wilt in a wide range of host plants (Hayward 1994). Safni et al. (2014) revised the taxonomy of the Ralstonia solanacearum species complex, describing the new species Ralstonia pseudosolanacearum, which encompassed the diversity of the previously recognised phylotypes I and III of Ralstonia solanacearum. Over 200 plant species are susceptible to these races of the pathogen, with the most economically important hosts in the family Solanaceae (Hayward 1994; Kelman 1953). Symptoms of infection are often of a vascular wilt, with bacterial streaming or ooze from a cut stem section being the most common clue to a presumptive diagnosis (Kelman 1953). Higher temperatures (24–35 °C) favour the disease, as does wet weather and high soil moisture levels (Nesmith and Jenkins 1985).
DNA was extracted from pure bacterial isolates using a Sigma REDExtract-N-Amp Plant PCR Kit. The 16S rDNA region was amplified using the fD1 and rP2 PCR primers. The endoglucanase egl gene was amplified using the primer pair Endo-F (5′-ATGCATGCCGCTGGTCGCCGC-3′) and Endo-R (5′-GCGTTGCCCGGCACGAACACC-3′). PCRs were performed in an Applied Biosystems Veriti Thermal Cycler in a total volume of 25 μL. The PCR mixtures contained 12.8 μL of UV-sterilised ultra-filtered water, 2.5 μL of 10× PCR buffer (with 20 mM MgCl2), 5.0 μL GC-RICH solution, 0.5 μL of dNTPs (each 100 μM), 1 μL of each primer (5 μM), 1 μL of BSA (10 μg/μl), 1 μL of genomic DNA (100 ng/μl), and 0.2 μL (1 U) of Roche FastStart Taq DNA Polymerase. The PCR conditions for 16S were 4 min at 94 °C, then 30 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 40 s, and then 7 min at 72 °C. The conditions for the egl gene were 4 min at 94 °C, then 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 40 s, and then 7 min at 72 °C. DNA sequences were obtained in both directions on an Applied Biosystems 3500xl.
A pathogenicity test was undertaken using six-week-old tomato seedlings from an organic seed farm in Paksong. The seedlings were transplanted into a soil mix consisting of two parts (by volume) of an alluvial soil that had no history of vegetable cropping, and one part sand, in small pots (15 cm diam), and grown at air temperatures varying from 25 to 30 °C. The alluvial soil was selected in order to provide a competitive soil microflora. There were four plants per pot. Two weeks after transplanting, cultures of ICMP 20038 were used to prepare suspensions of bacterial cells at a concentration of approximately 5 × 106 to 5 × 107 CFU ml−1 in sterile water. In the first test, plants were inoculated by pouring 10 ml of suspension per plant down the stem of the plant, starting approximately 10 cm from the growing tip of the plant. In this experiment, four plants (i.e. one pot) were wounded with a sterile hypodermic needle along the stem, below the inoculation point, while four plants were not. In the second test, a bacterial suspension made from re-isolations from the previous test was injected carefully into stems of plants until a small amount of inoculum exuded from the inoculation point. Sterile water was used to inoculate four plants in each test as a control. Once inoculated, the plants were covered with a moistened plastic bag for 24 hr to aid infection with a high relative humidity environment.
This is the first report of R. pseudosolanacearum in the Lao PDR. Symptoms consistent with bacterial wilt have also been observed in chilli and ginger at the large polyhouse farm near Paksong from where the samples of tomato were collected, indicating the possible presence of additional hosts of R. pseudosolanacearum in this region and potentially other phylotypes of R. pseudosolanacearum and closely related species. Furthermore, symptoms consistent with bacterial wilt have also been observed in tomato in small-holder farms in Savannakhet city, a more northern location at close to sea level (145 m alt) and different agro-climatic region to the Bolaven plateau. The production of tomatoes on R. pseudosolanacearum and R. solanacearum (sensu lato) resistant root stock is being encouraged, and is already being applied by some larger farming cooperatives in the area. This is a highly technical task however, and will require targeted and sustained capacity building efforts to reach a wide range of both smallholder and commercial-scale farms. In the meantime, capacity building programs to encourage clean planting stock, farm hygiene (“come clean, go clean”), adequate destruction of infected crop residues and crop rotation is ongoing. Additional work to identify the full range of R. solanacearum (sensu lato) phylotypes and distinct species in the region, coupled with the testing of suitable rotation vegetable crops for tolerance and resistance, when assessed alongside the growing knowledge of plant pathogens known to cause pathologies on the plateau (Callaghan et al. 2016; Ireland et al. 2014) would be valuable to inform and assist future cropping options and integrated disease management strategies.
Financial support from the Crawford Fund of Australia is gratefully acknowledged. The authors also acknowledge the support provided by the Champasak Provincial Agriculture and Forestry Office, and the Plant Protection Centre in Vientiane. The first author was an Australian Volunteer for International Development, the eleventh author was an Australian Youth Ambassador for Development, both Australian Government Programs, and the twelfth author was a Voluntary Service Overseas volunteer, a charity operating out of offices in the United Kingdom.
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