Background: Plant growth-promoting rhizobacteria (PGPR) are highly promising biofertilizers that contribute to eco-friendly sustainable agriculture. There have been many reports on the anti-microbial properties of nanoparticles (NPs). Toxic effects of NPs under laboratory conditions have also reported; however, there is a lack of information about their uptake and mobility in organisms under environmental conditions. There is an urgent need to determine the highest concentration of NPs which is not detrimental for growth and proliferation of PGPR.
Methods: Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to measure the size and shape of NPs. Minimum inhibitory concentrations (MIC) of nano-silver on selected beneficial microbes and Ralstonia solanacearum were measured using the microdilution broth method. The percentage of seed germination was measured under in vitro conditions.
Results: NPs were spherical with a size of 16 ± 6 nm. Nano-silver at 12-40 mg l-1 inhibited the growth of bacteria. Seed application at 40 mg l-1 protected seeds from R. solanacearum and improved the rate of seed germination.
Ralstonia solanacearum species complex phylotype IV strains, which have been primarily isolated from Indonesia, Australia, Japan, Korea, and Malaysia, have undergone recent taxonomic and nomenclatural changes to be placed in the species Ralstonia syzygii. This species contains three subspecies; Ralstonia syzygii subsp. syzygii, a pathogen causing Sumatra disease of clove trees in Indonesia, Ralstonia syzygii subsp. indonesiensis, the causal pathogen of bacterial wilt disease on a wide range of host plants, and Ralstonia syzygii subsp. celebesensis, the causal pathogen of blood disease on Musa spp. In Indonesia, these three subspecies have devastated the cultivation of susceptible host plants which have high economic value. Limited knowledge on the ecology and epidemiology of the diseases has hindered the development of effective control strategies. In this review, we provide insights into the ecology, epidemiology and disease control of these three subspecies of Ralstonia syzygii.
During March 2011 to June 2012, 50 banana plants of cultivar Musa × paradisiaca 'Horn' with Moko disease symptoms were randomly sampled in 12 different locations of 5 outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan, and Johor, with disease incidence exceeding 90% in some severely affected plantations. The disease symptoms observed in the infected plants included yellowing and wilting of the oldest leaves, which became necrotic, and eventually led to their dieback or collapse. The pulp of banana fruits also became discolored and exuded bacterial ooze. Vascular tissues in pseudostems were discolored. Fragments from symptomatic plant samples were excised and cultured on Kelman's-tetrazolium salt (TZC) medium. Twenty positive samples produced fluidal colonies that were either entirely white or white with pink centers after incubation for 24 to 48 h at 28°C on Kelman's-TZC medium and appeared as gram-negative rods after Gram staining. They were also positive for potassium hydroxide (KOH), Kovacs oxidase, and catalase tests, but negative for utilization of disaccharides and hexose alcohols, which are characteristics of biovar 1 Ralstonia solanacearum. For the pathogenicity test, 30 μl of 108 CFU/ml bacterial suspension of three selected virulent strains were injected into banana (Musa × paradisiaca 'Horn') leaves explants grown in plastic pots of 1,440 cm3 volume in a greenhouse, with temperature range from 26 to 35°C. Leaves that were infiltrated with sterile distilled water served as a negative control. Inoculations with all isolates were performed in three replications, as well as the uninoculated control leaves explants. The inoculated plants produced the same symptoms as observed on naturally diseased samples, whereas control plants remained asymptomatic. Strain cultures were re-isolated and possessed the morphological and biochemical characteristics as previously described. PCR amplification using race 2 R. solanacearum primers ISRso19-F (5'-TGGGAGAGGATGGCGGCTTT-3') and ISRso19-R (5'-TGACCCGCCTTTCGGTGTTT-3') (3) produced a 1,900-bp product from DNA of all bacterial strains. BLAST searches resulted that the sequences were 95 to 98% identical to published R. solanacearum strain race 2 insertion sequence ISRso19 (GenBank Accession No. AF450275). These genes were later deposited in GenBank (KC812051, KC812052, and KC812053). Phylotype-specific multiplex PCR (Pmx-PCR) and Musa-specific multiplex PCR (Mmx-PCR) were performed to identify the phylotype and sequevar of all isolates (4). Pmx-PCR showed that all isolates belonged to phylotype II, whereas Mmx-PCR showed that they belonged to phylotype II sequevar 4 displaying 351-bp amplicon. Although there were previously extensive studies on R. solanacearum associated with bacterial wilt disease of banana crops in Malaysia, none related to Moko disease has been reported (1,2). The result has a great importance to better understand and document R. solanacearum race 2 biovar 1, since banana has been identified as the second most important commercial fruit crop with a high economic value in Malaysia. References: (1) R. Khakvar et al. Plant Pathol. J. 7:162, 2008. (2) R. Khakvar et al. Am. J. Agri. Biol. Sci. 3:490, 2008. (3) Y. A. Lee and C. N. Khor. Plant Pathol. Bull. 12:57, 2003. (4) P. Prior et al. Pages 405-414 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. The American Phytopathological Society, St. Paul, MN, 2005.
The WRKY transcription factors (TFs) network is composed of WRKY TFs' subset, which performs a critical role in immunity regulation of plants. However, functions of WRKY TFs' network remain unclear, particularly in non-model plants such as pepper (Capsicum annuum L.). This study functionally characterized CaWRKY30-a member of group III Pepper WRKY protein-for immunity of pepper against Ralstonia solanacearum infection. The CaWRKY30 was detected in nucleus, and its transcriptional expression levels were significantly upregulated by R. solanacearum inoculation (RSI), and foliar application ethylene (ET), abscisic acid (ABA), and salicylic acid (SA). Virus induced gene silencing (VIGS) of CaWRKY30 amplified pepper's vulnerability to RSI. Additionally, the silencing of CaWRKY30 by VIGS compromised HR-like cell death triggered by RSI and downregulated defense-associated marker genes, like CaPR1, CaNPR1, CaDEF1, CaABR1, CaHIR1, and CaWRKY40. Conversely, transient over-expression of CaWRKY30 in pepper leaves instigated HR-like cell death and upregulated defense-related maker genes. Furthermore, transient over-expression of CaWRKY30 upregulated transcriptional levels of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. On the other hand, transient over-expression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 upregulated transcriptional expression levels of CaWRKY30. The results recommend that newly characterized CaWRKY30 positively regulates pepper's immunity against Ralstonia attack, which is governed by synergistically mediated signaling by phytohormones like ET, ABA, and SA, and transcriptionally assimilating into WRKY TFs networks, consisting of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. Collectively, our data will facilitate to explicate the underlying mechanism of crosstalk between pepper's immunity and response to RSI.