First Report of Ralstonia pseudosolanacearum Causing Wilt Disease in Tomato (Solanum lycopersicum L.) Plants from Mexico.

Mexico produces more than four million tons of tomato fruits and ranks tenth worldwide. In February 2022, tomato plants in a greenhouse in Culiacan, Sinaloa State, were affected by wilt diseases with an incidence of 20% and irreversible wilt and death of the infected plants (severity up 70%). When cut stems from affected plants, a reddish to brown discoloration of the vascular system was observed and these were disinfected with 1% NaClO for 5 min and then placed in a humid chamber. Characteristic milky-white exudate was obtained. From that exudate, irregular, mucoid, and white colonies with pink centres were obtained on casamino peptone glucose (CPG) plates supplemented with 1% 2,3,5-triphenyl 15 tetrazolium chloride (TZC); these characteristics are typical of the Ralstonia solanacearum species complex (RSSC) (Garcia et al., 2019). Identification of the pathogen was done by PCR using specific primer pairs reported by Paudel et al. (2022), RssC-wF3 (5'-TATATATCCTCGACTTTTCCATGAAGCTGTG-3') - RssCwR3 (5'-CTATATATATACCCCACTTGTTGAGGAACTG-3') and Rpseu-wF5 (5'-TTTTATTTTTTTGGTGTCCGGGCCAAGATAG-3') - Rpseu-wR5 (5'- TTATATTACTCGAACGTGCTGCAAAACCACT-3'), which amplified fragments of 162 and 251 bp for RSSC and Ralstonia pseudosolanacearum, respectively. Additionally, 759 (5'-GTCGCCGTCAACTCACTTTCC-3') - 760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') (Opina, et al., 1997) and Nmult21:1F (5'-CGTTGATGAGGCGCGCAATTT-3') - Nmult22:RR (5'- TCGCTTGACCCTATAACGAGTA-3') (Fegan and Prior, 2005) were used to generate 282 and 144 bp amplicons for RSSC and phylotype I, respectively. Subsequen to making the specific detection, the representative strain ClnMx was used to generate a sequence for the endoglucanase (egl) gene for separation into sequevars by using the primers Endo-F (5'- ATGCATGCCGCTGGTCGCCGC-3') and Endo-R (5'-GCGTTGCCCGGCACGAACACC-3'), which amplified a fragment of 750 bp (Fegan et al., 1998). The egl sequence (GenBank Access ON542479) showed 100% identity with the well-defined R. pseudosolanacearum sequevar 14, which was isolated from tomato plants from Senegal (UW763, I-14 GenBank Access CP051174) (Steidl et al., 2021), as well as, the strain MAFF 301070 (GenBank Access AB508612) from Japanese tomato. For pathogenicity tests, four 1-month-old tomato plants were infected using an insulin syringe that contained a pure bacterial suspension with approximately 2x108 CFU/mL. For each plant, 20 µL was infiltrated into the axil of the third upper leaf, and for untreated controls, tomato plants were infiltrated with sterile water. All plants were kept at 28°C under greenhouse conditions. Symptoms resembling those observed in the field were observed in inoculated plants six days after inoculation, and the plant pathogen was recovered on TZC medium. To confirm the bacteria identification a PCR using the specific primer pairs mentioned early was carried out. In contrast, water-treated control plants remained healthy. Koch's postulates were carried out twice with similar results. Ralstonia solanacearum species complex (RSSC) causes severe economic losses in many countries of the world because of their capability to infect a wide range of host plants, including potato, tomato, eggplant, tobacco, and, banana, among others. Ralstonia pseudosolanacearum has been reported to cause tomato wilt disease mainly on the Afro-Eurasian continent in areas such as Senegal, Cambodia, and Japan (Klass et al., 2019). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt diseases in tomato plants from Mexico and because, the control of this bacteria is a challenge by the long survival time in soil, water, and infected plant tissues, the identification of this important pathogen could provide relevant information for developing management strategies.

Fol-milR1, a pathogenicity factor of Fusarium oxysporum, confers tomato wilt disease resistance by impairing host immune responses.

Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection. Here, using sRNA-seq, we demonstrate that Fol-milR1, a trans-kingdom small RNA, is exported into tomato cells after infection. The knockout strain ∆Fol-milR1 displays attenuated pathogenicity to the susceptible tomato cultivar 'Moneymaker'. On the other hand, Fol-milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar 'Motelle'. Several tomato mRNAs are predicted targets of Fol-milR1. Among these genes, Solyc06g007430 (encoding the CBL-interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol-milR1. Furthermore, SlyFRG4 loss-of-function alleles created using CRISPR/Cas9 in tomato ('Motelle') exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol-milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus-induced gene silenced (VIGS) knock-down SlyAGO4a plants exhibit reduced susceptibility to Fol. Together, our findings support a model in which Fol-milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection.

Effect of Trichoderma spp. on Fusarium wilt disease of tomato.

Seven isolates of Trichoderma spp. (T1 to T7) from Egypt were evaluated in vitro by bioassay for their potential to antagonize Fusarium oxysporum f.sp. lycopersici (FOL, the causal pathogen of tomato wilt disease). The highest percentage of inhibition against the tested pathogenic isolate were obtained with Trichoderma isolate (T7) followed by Trichoderma isolate (T3). In greenhouse experiments, the application of the highly antagonistic isolates of Trichoderma spp. (T3 and T7) led to a significant decrease of disease severity compared to the untreated control treatment. The lowest severity was achieved with the T3 isolate (24.8%) followed by isolate T7 (34.6%) compared with the other tested isolates. To understand the ability of Trichoderma isolates to protect against wilt disease, its induced systemic resistance in tomato plants has been studied. The expression of a defense-related gene (ß-1,3-glucanase gene) was assessed by real-time RT-PCR in tomato plants to test the accumulation kinetics of transcripts encoding PR proteins in the roots of tomato in control (only with the pathogen), T3&FOL, and T7&FOL treatments. The highest degree of gene expression was found in tomato plants which were treated with T3&FOL compared with control (pathogen only). Two species of antagonistic Trichoderma (T3& T7) were characterized based on molecular tools using internal transcribed spacers (ITS1 and ITS4). The results of genetic characterization identified two different species of Trichoderma (T. atroviride and T. longibrachiatum).