Monika Jain

Ralstonia pseudosolanacearum ( Rps ) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 10 ⁹ CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::Ω mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::Ω . It indicated that the necrotic lesion caused in tomato leaves was hrp -dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Transformation of foreign DNA, subjecting Escherichia coli to CaCl₂ treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its popularity in E. coli, heat shock transformation is rarely reported in other Gram-negative bacteria. The objective of this research is to demonstrate that the heat shock transformation method can also be performed to introduce genes in different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii. A green fluorescent plasmid, pDSK-GFPuv, having the size of ~ 8.9 kb, was directly transferred to R. pseudosolanacearum by heat shock at 50 °C for 60s. For P. aeruginosa, P. putida, and E. roggenkampii, the cells were made competent using CaCl_2, followed by heat shock transformation at 50 °C for 180s. As the plasmid contains a gene encoding green fluorescence protein, after successful transformation, bacterial strains were demonstrated for colonization in tomato seedlings. The introduced plasmid was re-isolated from R. pseudosolanacearum to prove its stability inside the bacterium after the heat shock transformation. This easy and convenient method of transformation may be used for other Gram-negative bacteria to prove its wider applicability.

Transformation of foreign DNA, subjecting Escherichia coli to CaCl ₂ treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its large popularity in E. coli , heat shock transformation is rarely reported in other Gram-negative bacteria. Instead, techniques such as natural transformation, conjugation, or electroporation are used in those bacteria. In this study, we have successfully implemented the heat shock transformation in four different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii . The standard heat shock transformation procedure used for E. coli DH5α has been modified. The pDSK-GFPuv plasmid bearing the green fluorescence gene was directly transferred to Ralstonia pseudosolanacearum by heat shock at 50 ºC for 60 seconds. For Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii , the cells were made competent using CaCl _2, followed by performing transformation by heat shock at 50 ºC for 180 seconds. The transformants were resistant to kanamycin as well as exhibited fluorescence. These transformants were used to study the colonization pattern of tomato seedlings. Our study suggested that the heat shock transformation method can also be performed to introduce genes in other Gram-negative bacteria, other than E. coli .

Endophytes are a class of non-pathogenic microorganisms that reside within a plant and contribute to their health. Here, we isolated several endophytes from cotyledon-stage tomato seedlings that inhibited the growth of Ralstonia pseudosolanacearum , the bacterial wilt pathogen of tomato. One such endophyte, Pseudomonas aeruginosa SPT08, protected tomato seedlings as well as grown-up plants from the wilt disease. SPT08 also improved the tomato plant height by 20% and root growth by 60% in weight. SPT08 colonization inside tomato seedlings as well as in grown-up plants was studied using green fluorescent protein to demonstrate its endophytic behavior. SPT08 exhibited twitching and swimming motility and produced extracellular enzymes such as pectinase, protease, and amylase. SPT08 tested positive for several plant growth-promoting features such as phosphate solubilization, production of siderophore, plant hormone auxin, hydrogen cyanide, and ammonia. These features were further corroborated with SPT08 whole genome sequence. SPT08 genome is 6265489 bp, with 66.59% G+C and 5786 coding genes, including type II, III, and VI protein secretion systems. The antiSMASH tool identified potential for several secondary metabolites, including antibiotics, in SPT08. This study underscores the utility of P. aeruginosa SPT08 bio-protection from bacterial wilt and growth promotion agent in tomato. Highlights: Pseudomonas aeruginosa SPT08, an endophyte isolated from tomato seedlings that inhibits bacterial wilt pathogen R. pseudosolanacearum, can control bacterial wilt in tomato plants and enhance the height and root growth.

Accurate enumeration of bacteria in a culture is the first step in both fundamental as well as applied research in microbiology. Serial dilution is an age old method used widely by researchers for enumerating viable bacteria in a culture where a specific sample volume is passaged successively to a specific diluent volume. Here, we demonstrated that a higher sample volume is a better representation of bacterial population than a lower sample volume, which was in concordance with the random nature of bacterial distribution in culture. Therefore, a bigger sample to diluent ratio during serial dilution appears more favorable for an accurate bacterial enumeration than a smaller ratio. But surprisingly, enumeration using the different dilution ratios such as 1:9, 1:99 and 1:999 in 1.0 mL final volume yielded similar results with the exception of 1:999, where 1 μL sample was passaged. However, in 10.0 mL final volume of dilution, the above three dilution ratios exhibited similar bacterial enumeration. The experiment was performed using two different bacterial cultures such as Escherichia coli and Ralstonia pseudosolanacearum . Our results indicated that the advantage gained due to lesser number of passages in case of a lower sample volume could overcome the disadvantage associated with it, thereby co-aligning the different dilution ratios with regards to enumeration. Hence, although in laboratory, 1:9 dilution ratio is usually performed during serial dilution, our results suggest that dilution ratios such as 1:99 in 1 mL dilution volume and ratios such as 1:99 and 1:999 in 10 mL dilution volume are equally effective, which also reduces time, cost and labor.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plant species, posing significant economic challenges. Although tomato has been a primary model host for investigating the pathogenicity and systemic infection of this bacterium, this manuscript presents a comparative pathogenicity study between two closely related solanaceous hosts, tomato and eggplant, revealing differential host responses to the same pathogen, specifically through leaves and roots. Interestingly, eggplant seedlings exhibited a significantly higher susceptibility to cotyledon leaf inoculation than tomato seedlings. In the case of tomato, a few seedlings escaped wilting (called escapees), which was usual at a high pathogen load, unlike the case of eggplant seedlings where escapees could not be observed even at a 100-fold lower pathogen concentration. The greater susceptibility of eggplant was further demonstrated by performing both single- and double-leaf inoculations in the seedlings. Surprisingly, root inoculations resulted in a significantly lower mortality for eggplant than for tomato seedlings. The contrasting susceptibility between the two hosts regarding root and leaf regions indicates the tissue-dependent nature of susceptibility. The study underscores the value of employing multiple host species to uncover new insights into pathogen behaviour and host-pathogen interactions.