Graphical abstract

This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen.

Aims: To understand the factors that contribute to the variations in colonyforming units (CFU) in different bacteria during spread plating. Methods and Results: Employing a mix culture of vegetative cells of ten organisms varying in cell characteristics (Gram reaction, cell shape and cell size), spread plating to the extent of just drying the agar surface (50-60 s) was tested in comparison with the alternate spotting-and-tilt-spreading (SATS) approach where 100 ll inoculum was distributed by mere tilting of plate after spotting as 20-25 microdrops. The former imparted a significant reduction in CFU by 20% over the spreader-independent SATS approach. Extending the testing to single organisms, Gram-negative proteobacteria with relatively larger cells (Escherichia, Enterobacter, Agrobacterium, Ralstonia, Pantoea, Pseudomonas and Sphingomonas spp.) showed significant CFU reduction with spread plating except for slow-growing Methylobacterium sp., while those with small rods (Xenophilus sp.) and cocci (Acinetobacter sp.) were less affected. Among Grampositive nonspore formers, Staphylococcus epidermidis showed significant CFU reduction while Staphylococcus haemolyticus and actinobacteria (Microbacterium, Cellulosimicrobium and Brachybacterium spp.) with small rods/cocci were unaffected. Vegetative cells of Bacillus pumilus and B. subtilis were generally unaffected while others with larger rods (B. thuringiensis, Brevibacillus, Lysinibacillus and Paenibacillus spp.) were significantly affected. A simulated plating study coupled with live-dead bacterial staining endorsed the chances of cell disruption with spreader impaction in afflicted organisms. Conclusions: Significant reduction in CFU could occur during spread plating due to physical impaction injury to bacterial cells depending on the spreader usage and the variable effects on different organisms are determined by Gram reaction, cell size and cell shape. The inoculum spreader could impart physical disruption of vegetative cells against a hard surface. Significance and Impact of Study: Possibility of CFU reduction in sensitive organisms and the skewed selection of hardier organisms during spread plating, and the recommendation of SATS as an easier and safer alternative for CFU enumerations.

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 108 CFU/ml−1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.

Evaluating different swabbing materials for spore recovery efficiency (RE) from steel surfaces, we recorded the maximum RE (71%) of 10 7 Bacillus subtilis spores with Tulips cotton buds, followed by Johnson's cotton buds and standard Hi-Media cotton, polyester, nylon, and foam (23%) swabs. Among cotton swabs, instant water-absorbing capacity or the hydrophilicity index appeared to be the major indicator of RE, as determined by testing three more brands. Tulips swabs worked efficiently across diverse nonporous surfaces and on different Bacillus spp., registering 65 to 77% RE. P roper sampling and retrieval methods are important for the surveillance of spores of hazardous pathogens, like Bacillus anthracis, and for monitoring microbial populations in space research in addition to traditional applications in food, clinical, and general microbiology (1-3). While wipes and vacuum suction are considered ideal for large-area sampling (1), swabs are preferred for small-area monitoring (4-6). Our prime interest in monitoring spore load on nonporous surfaces was directed at increasing our preparedness to address accidental surface contamination from different Bacillus spp. (7) in order to ensure a clean working environment.The spore recovery efficiency (RE) in past studies employing swabs varied depending on the surface sampled and the swabbing material, with most of the studies generally reporting Ͻ50% RE (2-5). In addition, little information has appeared to be available from developing parts of the world with regard to effective spore surveillance, the input of which would become valuable in the event of an unprecedented public health hazard arising from the dreaded B. anthracis. Although different compositions of swabs, such as cotton, foam, polyester, rayon, sponge, and blends, are available commercially, none has been found to be universally acceptable (1,(3)(4)(5)(6). Cotton swabs are easily available worldwide and have registered higher RE than synthetic swabs in some studies (6). Further, we also felt it prudent to try the universally available cotton buds (also called ear buds or Q-tips) for spore surveillance. This study was undertaken to develop an efficient spore surveillance methodology applicable across different surfaces and organisms.B. subtilis (ATCC 6051) was used as the primary test organism. A spore suspension prepared from 7-to 10-day-old nutrient agar (NA) cultures (30°C) in sterile distilled water (DW) after 70°C heat treatment (10 min) was dispersed in 50% ethanol, and the optical density at 600 nm (OD) was adjusted to 2.0. The spore suspension showed an initial CFU of 1.26 ϫ 10 9 to 1.43 ϫ 10 9 ml Ϫ1 , which after overnight storage dropped to 1.02 Ϯ 0.189 ϫ 10 9 ml Ϫ1 but thereafter remained consistent with 4°C storage over 8 weeks of monitoring.Different standard swab materials from Hi-Media (HM) Biosciences (Mumbai, India), designated HM-foam, HM-nylon, HM-polyester, and HM-cotton, and two brands of cotton buds, namely Johnson's (Johnson & Johnson, manufactured at Mumbai, India) and Tulips (M/s...