Pseudomonas syringae and Botrytis cinerea cause destructive bacterial speck and grey mold diseases in many plant species, leading to substantial economic losses in agricultural production. Our study discovered that the application of Bacillus proteolyticus strain OSUB18 as a root-drench enhanced the resistance of Arabidopsis plants against P. syringae and B. cinerea through activating Induced Systemic Resistance (ISR). The underlying mechanisms by which OSUB18 activates ISR were studied. Our results revealed that the Arabidopsis plants with OSUB18 root-drench showed the enhanced callose deposition and ROS production when inoculated with Pseudomonas syringae and Botrytis cinerea pathogens, respectively. Also, the increased salicylic acid (SA) levels were detected in the OSUB18 root-drenched plants compared with the water root-drenched plants after the P. syringae infection. In contrast, the OSUB18 root-drenched plants produced significantly higher levels of jasmonyl isoleucine (JA-Ile) than the water root-drenched control after the B. cinerea infection. The qRT-PCR analyses indicated that the ISR-responsive gene MYC2 and the ROS-responsive gene RBOHD were significantly upregulated in OSUB18 root-drenched plants upon both pathogen infections compared with the controls. Also, twenty-four hours after the bacterial or fungal inoculation, the OSUB18 root-drenched plants showed the upregulated expression levels of SA-related genes (PR1, PR2, PR5, EDS5, and SID2) or JA-related genes (PDF1.2, LOX3, JAR1 and COI1), respectively, which were consistent with the related hormone levels upon these two different pathogen infections. Moreover, OSUB18 can trigger ISR in jar1 or sid2 mutants but not in myc2 or npr1 mutants, depending on the pathogen’s lifestyles. In addition, OSUB18 prompted the production of acetoin, which was reported as a novel rhizobacterial ISR elicitor. In summary, our studies discover that OSUB18 is a novel ISR inducer that primes plants’ resistance against bacterial and fungal pathogens by enhancing the callose deposition and ROS accumulation, increasing the production of specific phytohormones and other metabolites involved in plant defense, and elevating the expression levels of multiple defense genes.

Plant diseases caused by the pathogen Pseudomonas syringae are serious problems for various plant species worldwide. Accurate detection and diagnosis of P. syringae infections are critical for the effective management of these plant diseases. In this review, we summarize the current methods for the detection and diagnosis of P. syringae, including traditional techniques such as culture isolation and microscopy, and relatively newer techniques such as PCR and ELISA. It should be noted that each method has its advantages and disadvantages, and the choice of each method depends on the specific requirements, resources of each laboratory, and field settings. We also discuss the future trends in this field, such as the need for more sensitive and specific methods to detect the pathogens at low concentrations and the methods that can be used to diagnose P. syringae infections that are co-existing with other pathogens. Modern technologies such as genomics and proteomics could lead to the development of new methods of highly accurate detection and diagnosis based on the analysis of genetic and protein markers of the pathogens. Furthermore, using machine learning algorithms to analyze large data sets could yield new insights into the biology of P. syringae and novel diagnostic strategies. This review could enhance our understanding of P. syringae and help foster the development of more effective management techniques of the diseases caused by related pathogens.

Trichoderma is a genus of wood-decaying fungi generally found in soil (Druzhinina and Kubicek 2005). Trichoderma crassum was confirmed to be a sister species to T. virens according to the molecular sequencing results (Chaverri et al. 2003). A foliar disease with ~70% incidence on Solanum lycopersicum was observed in a greenhouse at The Ohio State University (40°0’8’’ north latitude, 83°1’36’’ west longitude), Columbus, United States, in December 2021. On average up to 60% of the leaves per two-month-old tomato plant were infected. Initially, the dark-grey color and irregular spots appeared at the leaf tips. As the disease progressed, the yellow necrotic lesions were observed surrounding the preformed disease spots. Finally, the infected leaves appeared curled and wilted as a whole. The leaf fragments from three tomato plants 40 inches apart were cut from the diseased lesions and surface sterilized with 75% ethanol (30 seconds) and 1% NaOCl (60 seconds), subsequently rinsed with sterilized deionized water three times. Nine pieces of the sterilized leaf tissues were then placed on the PDA plates at 28℃ in the dark and incubated in one incubator for 4 days. The pure cultures of five isolates were acquired and examined with a light microscope. The fungus from all the isolates changed from white to dark green with the radial pattern and profuse sporulation on the PDA. The produced round conidia were observed under a light microscope (Fig S1). The DNA was extracted from two representative isolates which showed the same morphology. The internal transcribed spacer (ITS) region and a conserved fungal rRNA region were amplified using the primers ITS1/ITS4 (5’-TCCGTAGGTGAACCTGCGG-3’ and 5’-TCCTCCGCTTATTGATATGC-3’) (White et al. 1990) and SR6f/SR7r (5’-TGTTACGACTTTTACTT-3’ and 5’-AGTTAAAAAGCTCGTAGTTG-3’) (Hirose et al. 2012), respectively. The PCR products were further sequenced by Sanger sequencing (Table S1). Based on the BLAST results through NCBI website, the ITS sequences of the two isolates were 99% (566/572) and 98% (558/572) identical to Trichoderma crassum DAOM 164916 (EU280067). Their SR sequences both showed 99% (290/293; 289/293) identity to the same strain. The phylogenetic tree was also created with the sequences of ITS region by MEGA software (version 11) (Fig S2). Therefore, the fungus was identified as Trichoderma crassum based on its morphological characteristics (green conidia), Sanger sequencing results, and phylogenetic tree. To complete Koch’s postulates, the 5-mm-diameter fungal agar discs of 7-day-old pure cultures were used for the inoculation on 18 healthy leaves of six tomato cv. M82 plants with two-month-old. The sterile pure PDA discs of the equal size were used for the mock inoculation as a comparison. Fungal plug method was chosen in this study because it had been widely applied to characterization of the fungal pathogens causing leaf spot disease (Pornsuriya et al. 2020; Yang et al. 2021). Five days later, the same symptom as those that occurred on the previously naturally infected tomato plants were observed on all the inoculated leaves (Fig S3A). However, there were no symptoms on the leaves with the mock inoculation. The fungus re-isolated from the symptomatic leaves showed the consistent morphology (dark-green color with radial sporulation) with the original isolates (Fig S3B). Thus, Trichoderma crassum was verified as the causal agent of the foliar disease on Solanum lycopersicum cv. M82 in our greenhouse. To our knowledge, it is the first report of Trichoderma crassum leading to the leaf spot and wilt on tomato in Ohio. The identification of the causal agent lays the groundwork for the development of necessary disease management techniques. We acknowledge the funding support from CFAES Internal Grants Program 2021009.

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