History of a model plant growth-promoting rhizobacterium, Bacillus velezensis GB03: from isolation to commercialization.

Bacillus velezensis strain GB03 is a Gram-positive rhizosphere bacterium known for its ability to promote plant growth and immunity. This review provides a comprehensive overview of the research on GB03 from its initial discovery in Australian wheat fields in 1971 to its current applications. Recognized as a model plant growth-promoting rhizobacterium (PGPR), GB03 has exhibited outstanding performance in enhancing the growth and protection of many crop plants including cucumber, pepper, wheat, barley, soybean, and cotton. Notably, GB03 has been reported to elicit plant immune response, referred to as induced systemic resistance (ISR), against above-ground pathogens and insect pests. Moreover, a pivotal finding in GB03 was the first-ever identification of its bacterial volatile compounds, which are known to boost plant growth and activate ISR. Research conducted over the past five decades has clearly demonstrated the potential of GB03 as an eco-friendly substitute for conventional pesticides and fertilizers. Validating its safety, the U.S. Environmental Protection Agency endorsed GB03 for commercial use as Kodiak® in 1998. Subsequently, other compounds, such as BioYield™, were released as a biological control agent against soil-borne pathogens and as a biofertilizer, utilizing a durable spore formulation. More recently, GB03 has been utilized as a keystone modulator for engineering the rhizosphere microbiome and for eliciting microbe-induced plant volatiles. These extensive studies on GB03 underscore its significant role in sustainable agriculture, positioning it as a safe and environmentally-friendly solution for crop protection.

Integrated Effect of Plastic Mulches and Biorational Insecticides in Managing Tomato Chlorotic Spot Virus (TCSV) and Its Vector Thrips in Tomatoes.

In the USA, tomato chlorotic spot virus (TCSV) was first identified in Miami-Dade County of Florida in 2012. This viral disease is transmitted by thrips (Thysanoptera: Thripidae) of different species, imposing a serious threat to the entire tomato production in the state. Both cultural and chemical control techniques could be essential tools to combat this vector-borne disease. In the present two-year-long study, we determined the effect of different types of plastic mulches and biorational insecticides on managing thrips and TCSV. Results from the leaf and flower samples showed a significantly lower adult thrips population in Entrust®SC treated tomatoes than in other treated and untreated tomatoes in 2018. Silver on black and silver on white reflective plastic mulches significantly reduced the adult thrips population in 2018. In both study years, marketable yield was significantly higher in tomatoes treated with Entrust®SC and reflective plastic mulches than in other treatments. The incidence of TCSV was significantly reduced in tomatoes treated with Entrust®SC and reflective plastic mulches than the untreated control in 2018. Marketable yield was negatively correlated with the thrips population, as observed from the Pearson correlation coefficient analysis. This research describes a potentially viable management program for thrips and thrips-transmitted TCSV.

Lentinan protects Caenorhabditis elegans against fluopyram-induced toxicity through DAF-16 and SKN-1 pathways.

Fluopyram, a SDH inhibitor fungicide, is widely used in agriculture to control fungi and nematodes. However, fluopyram has been proved toxic that caused damage to organs through oxidative stress. The development of natural extracts that can reduce oxidative damage is a promising method. Lentinan is isolated from Lentinus edodes and has been verified its antioxidant activity. In this study, Caenorhabditis elegans was used to evaluate the protective effects of lentinan against fluopyram-induced toxicity and the possible mechanisms. Results showed that lentinan pretreatment notably increased the survival rate of N2 nematodes by 15.0 % and extended the lifespan by 91.5 %, compared with the fluopyram treatment. Lentinan pretreatment reverted the inhibition of the locomotion and reproduction of C. elegans under the fluopyram stress. In addition, lentinan pretreatment significantly decreased the contents of ROS and MDA in N2 nematodes. Moreover, pretreated with lentinan significantly recovered the decreased activities of CAT, SOD, GST and SDH induced by fluopyram. Lentinan pretreatment enhanced the mRNA levels of daf-16 and skn-1 and their downstream genes in the nematodes compared with the fluopyram group. In daf-16 and skn-1 mutants, the lifespan, ROS and related genes expression were not significantly changed in lentinan pretreatment. Pretreated with lentinan significantly enhanced the fluorescence intensity of SOD-3::GFP and GST-4::GFP, and promoted the nuclear translocation of DAF-16 and SKN-1 under the fluopyram stress. In summary, these findings indicated that lentinan protected C. elegans from fluopyram-induced toxicity via DAF-16 and SKN-1.

First Report of Leaf Blight Caused by Curvularia aeria and C. senegalensis on Tomato (Solanum lycopersicum L.) in Florida, USA.

Tomato (Solanum lycopersicum L.) is an important economic crop in Florida and worldwide. In November 2021, a leaf blight was reported on tomato plants (hybrid cherry and artisan tomatoes) from a small farm in Miami-Dade County, Florida. About 100 plants showed symptoms with disease severity of 15% and disease incidence of 80%. Symptoms on the leaves started as small dark spots and coalesced to form larger necrotic lesions over time. Symptomatic leaf tissues were cut into 5-mm pieces, surface disinfected with 70% ethanol for 30 s and 1% NaClO for 5 min, then cultured on PDA for 3 to 5 days at 25°C. Isolations were conducted in three rounds, with 15 samples in each round. Except for the saprophytes, fungal isolates of Curvularia were consistently recovered from tissues in each round. Single spore isolates grouped in two morphotypes (CT1 and CT3, CT2 and CT4) were examined for morphological and molecular identification. Colonies on PDA were dark yellow-green, with a fluffy surface, then both morphotypes turned black, although CT2 and CT4 were light yellow at the edges. CT1 and CT3 produced light-brown, straight to curved conidia with smooth walls and 1 to 3 septa, 18 to 28 ✕ 9 to 12 µm (n=60), and dark-brown stromatic synnemata (> 200 µm in length) in the center of the colony after ~30 days of incubation in PDA. CT2 and CT4 produced brown, mostly curved conidia, 14 to 23 ✕ 8 to 9 µm (n=60), with slightly rugose walls and 3-4 septa, without synnemata. A dehydrated culture of each isolate was deposited in the Plant Industry Gainesville Herbarium [(PIGH, accession numbers 17443 (CT1), 17444 (CT2), 17445 (CT3), 17446 (CT4)]. Total DNA was extracted using DNeasy Plant Pro Kit (Qiagen, Germantown, MD) followed by PCR amplification and Sanger sequencing of the internal transcribed spacers (ITS) and the Large Subunit (LSU) of the rRNA gene, together with the protein coding gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1- (TEF1) and -tubulin (TUB) (Marin-Felix et al. 2020; Himashi et al. 2021; Manamgoda et al. 2012; Myllys et al. 2002) (GenBank accession numbers ITS-LSU: OQ657944-OQ657947, GAPDH: OQ689438 to OQ689441, TEF1: OQ689442 to OQ689445, TUB: OQ689446 to OQ689449). Curvularia clavata as the molecular marker (96% identity) was used for identification. Sequence similarity of 100% in GAPDH, ITS and LSU was obtained in megaBLAST searches for both groups of morphotypes, CT1 and CT3 to Curvularia aeria (Bat., J.A. Lima & C.T. Vasconc.) Tsuda type culture CBS 294.61, and CT2 and CT4 to Curvularia senegalensis (Speg.) Subram. culture CBS 149.71. Pathogenicity tests were conducted with each isolate on six tomato plants that were 6- weeks-old. The seeds used in the tests were provided by the farm, and the variety 'Red Bounty' was also used. Inoculation was accomplished by spraying a spore suspension (1 x 106 spores/ml) of each of the four isolates (CT1 to CT4) and by placing 6-mm PDA plugs of the isolates on the leaves. Six tomato plants were used as the control. All plants were covered by plastic bags and placed in a greenhouse at 23-27°C. The inoculated plants developed small dark spots on leaves 2 weeks after inoculation, and the leaves inoculated by plugs of the fungal isolates had large necrotic lesions, which were similar to those observed on tomato plants from the field. The pathogenicity tests were repeated three times, Curvularia was consistently isolated from inoculated leaves after the symptoms developed, and they were confirmed morphologically in each test. No symptoms were observed from the control plants. Curvularia aeria and C. senegalensis are known foliar pathogens on several important crops, but not tomatoes. To our knowledge, this is the first report of C. aeria and C. senegalensis causing leaf blight in tomatoes worldwide. This finding is important because it will extend the host range of C. aeria and C. senegalensis to tomato, it also implied the essentiality of crop rotation in disease management.

Oxidative stress and mitochondrial damage induced by a novel pesticide fluopimomide in Caenorhabditis elegans.

Fluopimomide is a novel pesticide intensively used in agricultural pest control; however, its excessive use may have toxicological effects on non-target organisms. In this study, Caenorhabditis elegans was used to evaluate the toxic effects of fluopimomide and its possible mechanisms. The effects of fluopimomide on the growth, pharyngeal pumping, and antioxidant systems of C. elegans were determined. Furthermore, the gene expression levels associated with mitochondria in the nematodes were also investigated. Results indicated that fluopimomide at 0.2, 1.0, and 5.0 mg/L notably (p < 0.001) decreased body length, pharyngeal pumping, and body bends in the nematodes compared to the untreated control. Additionally, fluopimomide at 0.2, 1.0, and 5.0 mg/L notably (p < 0.05) increased the content of malondialdehyde by 3.30-, 21.24-, and 33.57-fold, respectively, while fluopimomide at 1.0 and 5.0 mg/L significantly (p < 0.001) increased the levels of reactive oxygen species (ROS) by 49.14% and 77.06% compared to the untreated control. In contrast, fluopimomide at 1.0 and 5.0 mg/L notably reduced the activities of target enzyme succinate dehydrogenase and at 5.0 mg/L reduced the activities of antioxidant enzyme superoxide dismutase. Further evidence revealed that fluopimomide at 1.0 and 5.0 mg/L significantly inhibited oxygen consumption and at 0.2, 1.0, and 5.0 mg/L significantly inhibited ATP level in comparison to the untreated control. The expression of genes related to the mitochondrial electron transport chain mev-1 and isp-1 was significantly downregulated. ROS levels in the mev-1 and isp-1 mutants after fluopimomide treatments did not change significantly compared with the untreated mutants, suggesting that mev-1 and isp-1 may play critical roles in the toxicity induced by fluopimomide. Overall, the results demonstrate that oxidative stress and mitochondrial damage may be involved in toxicity of fluopimomide in C. elegans.

Biocontrol Efficacy of Bacillus methylotrophicus TA-1 Against Meloidogyne incognita in Tomato.

Root-knot nematodes (RKNs) are harmful plant-parasitic nematodes of tomatoes which can cause significant yield losses. Therefore, there is increasing interest in exploring the application of bacterial nematicides. The bacterium Bacillus methylotrophicus TA-1 is a broad-spectrum biological control agent; however, its effect on RKNs control remains largely unclear. In this study, the toxicity of B. methylotrophicus TA-1 against Meloidogyne incognita was investigated in vitro, and the potential of B. methylotrophicus TA-1 to decrease infection of RKNs in tomato were evaluated in pot and field trials. Results showed that B. methylotrophicus TA-1 exhibited high nematicidal activity against second-stage juveniles (J2s) and eggs of M. incognita with 50% lethal concentration (LC50) values of 5.80 and 7.00 × 108 colony forming units (CFU)/ml, respectively. In the pot experiments and field trials conducted in 2020 and 2021, tomato plants treated with B. methylotrophicus TA-1 soil drench applied once at 3, 6, and 9 × 108 CFU/plant had significantly higher plant height and greater yield compared with the untreated control. Tomato yields of the treated plots with B. methylotrophicus TA-1 in 2 consecutive years' field trials were between 53.4 to 66.1 and 52.8 to 61.5 t/ha, while they were 49.7 and 48.2 t/ha in the untreated control for each year, respectively. The lowest population densities of M. incognita at 30 and 60 days after treatment were 119 and 135 J2s per 100 g soil in 2020 and 43 and 118 J2s in 2021 in TA-1-treated plots. The lowest gall index of 4.7 and 3.3 in 2020 and 2021, respectively, and the highest yield were all observed in the TA-1 at 9 × 108 CFU/plant treated plants, with no significant differences with the commercial control abamectin. These results provided a basis for further studies of B. methylotrophicus TA-1 formulations, application doses, frequencies, and mechanisms of action, which are necessary before it could be used as a component of integrated management programs to manage RKNs in tomato production.

Evaluating Nicotinamide Adenine Dinucleotide for Its Effects on Halo Blight of Snap Bean.

Halo blight, caused by Pseudomonas syringae pv. phaseolicola, is one of the major bacterial diseases on snap bean in Florida, and the outbreaks of this disease have occurred more often in recent years. Current management of this disease primarily depends on application of fixed copper-based bactericides but climate change and resistance development in the pathogen populations still cause hardship for management of this disease, especially in south Florida. In this study, nicotinamide adenine dinucleotide (NAD+) was evaluated in the greenhouse for its potential to reduce halo blight on snap bean. When NAD+ at 5 mM was applied by soil drench, foliar spray, or leaf infiltration, NAD+ significantly (P < 0.05) reduced disease severity of halo blight on snap bean compared with the untreated control. When NAD+ was applied by leaf infiltration, among the tested concentrations, NAD+ at 0.5 to 1.0 mM was most effective in decreasing halo blight disease. NAD+ at 2.5 mM applied as a foliar spray in rotation with Kocide 3000 (copper hydroxide) at 0.5 mg/ml further reduced disease severity compared with Kocide 3000 alone. In the in vitro study, no inhibitory effects of NAD+ were detected on the bacterial pathogen P. syringae pv. phaseolicola. Results of real-time PCR showed that the defense-related genes PR1, AZI1, EDS1, SARD1, PDF1.2, and PAL1 were upregulated in the NAD+ treatment. Taken together, these data indicated that NAD+ significantly suppressed halo blight on snap bean, and application of NAD+ has the potential in management of this important disease.

Potential and Metabolic Pathways of Eugenol in the Management of Xanthomonas perforans, a Pathogen of Bacterial Spot of Tomato.

Bacterial spot of tomato continues to pose a significant problem to tomato production worldwide. In Florida, bacterial spot of tomato caused by Xanthomonas perforans is one of the most important diseases responsible for tomato yield loss. This disease is difficult to control, and new strategies are continually being investigated to combat the devastating effect of this disease. Recent efforts focusing on essential oils based on small molecules have spurred interests in the utilization of this class of chemicals for disease management. In this study, we evaluated the efficacy of eugenol for the management of bacterial spot of tomato caused by X. perforans. In the greenhouse experiments, eugenol applied as a foliar spray significantly (p < 0.5) reduced bacterial spot disease compared to the untreated control. In the field experiments, the area under the disease progress curve (AUDPC) was significantly (p < 0.5) lower in the plots treated with eugenol or eugenol combined with the surfactant Cohere than in the untreated control plots, and it was comparable to the copper-based treatments. To provide additional insights into the possible pathways of eugenol activities, we applied a liquid chromatography mass spectrometry (LC-MS)-based metabolomic study using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) on X. perforans strain 91−118 treated with eugenol. Our results showed that eugenol affected metabolite production in multiple pathways critical to bacterial survival. For example, treatment of cells with eugenol resulted in the downregulation of the glutathione metabolism pathway and associated metabolites, except for 5-oxoproline, which accumulation is known to be toxic to living cells. While the peaks corresponding to the putatively identified sarmentosin showed the most significant impact and reduced in response to eugenol treatment, branched-chain amino acids, such as L-isoleucine, increased in production, suggesting that eugenol may not negatively affect the protein biosynthesis pathways. The results from our study demonstrated the efficacy of eugenol in the management of bacterial spot of tomato under greenhouse and field conditions and identified multiple pathways that are targeted.

Exposure to fluopimomide at sublethal doses causes oxidative stress in Caenorhabditis elegans regulated by insulin/insulin-like growth factor 1-like signaling pathway.

Fluopimomide is an innovative pesticide, widely used for agricultural pest management; however, little is known about its effect on non-target organisms. This study was designed to assess the potential risk of fluopimomide and the molecular mechanisms using Caenorhabditis elegans, a common model animal. The oxidative stress-related indicators were analyzed in C. elegans after exposure to fluopimomide for 24 h at three sublethal doses (0.2, 1.0, and 5.0 mg/L). The results demonstrated that sublethal exposure to fluopimomide adversely affected the nematodes growth, locomotive behaviors, reproduction, and lifespan, accompanying with enhanced of reactive oxygen species (ROS) generation, lipid and lipofuscin accumulation, and malondialdehyde content. In addition, exposure to fluopimomide significantly inhibited antioxidant systems including superoxide dismutase, catalase, glutathione S-transferase, and glutathione in the nematodes. Moreover, the expression of oxidative stress-related genes of sod-3, hsp-16.1, gst-4, ctl-2, daf-16, and daf-2 were significantly down-regulated, while the expression of skn-1 was significantly up-regulated. Further evidence revealed that daf-16 and skn-1 mutant strains of C. elegans significantly decreased ROS production upon fluopimomide exposure compared with the wild-type nematodes. Overall, our findings indicated that exposure to fluopimomide at sublethal doses caused oxidative damage, mainly associated with insulin/IGF-1-like signaling pathway in C. elegans. This is the first report of potential toxic effects of fluopimomide even at low concentrations, providing a new insight into the mechanisms of toxicity to C. elegans by fluopimomide.

Lasiodiplodia iraniensis, a New Causal Agent of Tuber Rot on Yam (Dioscorea Species) Imported into the United States and Implications for Quarantine Decisions.

One negative consequence of international trade of agricultural commodities is the inadvertent global spread of crop diseases. Yam (Dioscorea spp.) is a staple food crop in many countries and is traded globally. Most of the commercially traded yams in the United States are imported. In late 2020, samples of yam tubers from a commercial facility were submitted to the plant diagnostic clinic at the UF/IFAS Tropical Research and Education Center in Homestead, Florida. Samples showed rotten symptoms and were drawn from lots that were marked to be destroyed because the source of the rotting symptoms was unknown. Preliminary isolation showed that a fungus was consistently associated with the symptoms and was confirmed in the subsequent pathogenicity test as the causal agent. The fungus grew profusely on potato dextrose agar (PDA) with highly melanized hyphae. Matured conidia showed longitudinal striations. Based on its growth pattern and morphology, it was suspected that this fungus may be in the genus Lasiodiplodia. DNA-based identification using partial sequences of the internal transcribed spacer (ITS), ß-tubulin (TUB2), 28S rDNA (LSU), and elongation factor alpha (EF1-α) genes confirmed the identity of the isolates as Lasiodiplodia iraniensis Abdollahz., Zare & A.J.L. Phillips (synonym: L. iranensis). This is the first report of L. iraniensis affecting yam and has implications for international trade. This finding will provide an important foundation for making quarantine decisions to prevent spread of this disease.

Reduced Sensitivity to Tebuconazole in Botryosphaeria dothidea Isolates Collected from Major Apple Production Areas of China.

China has the largest acreage and is the greatest producer of apples in the world. Apple ring rot (ARR) caused by Botryosphaeria dothidea is one of the major diseases affecting apple fruit production. Tebuconazole, a sterol demethylation-inhibiting (DMI) fungicide, has been increasingly applied to manage ARR in China. Resistance to tebuconazole in field populations of B. dothidea may be selected and accumulate to higher degrees over time. Establishment of tebuconazole sensitivity monitoring programs is critically important for resistance management and effective ARR control. However, the resistance level of B. dothidea to tebuconazole in China remains largely unknown. In this study, in vitro mycelial growth assays of B. dothidea in media amended with tebuconazole were conducted, and the sensitivity of B. dothidea to tebuconazole was determined with a set of 390 isolates collected from the major apple production provinces in China between 2006 and 2014. Results showed that the 50% effective concentration (EC50) value ranged from 0.011 to 0.918, 0.040 to 1.621, and 0.052 to 1.925 µg ml-1 with a median value of 0.194, 0.386, and 0.782 µg ml-1 in the isolates collected in 2006, 2010, and 2014, respectively. The frequency distribution of EC50 for tebuconazole was a nonnormal distribution (P < 0.05), suggesting that subpopulations with reduced sensitivity to tebuconazole had emerged in these B. dothidea isolates. The frequency distribution of the B. dothidea isolates collected in 2006 fit a unimodal curve and could be regarded as the baseline sensitivity to tebuconazole. The resistance levels increased over time with the average occurrence frequency of 43.3% and resistance index of 0.38. Positive cross-resistance was observed between tebuconazole and metconazole, which is another DMI fungicide, but multiple resistance was not detected between tebuconazole and non-DMI fungicides. Our results demonstrated that regular long-term resistance monitoring combining with prudent fungicide use should be implemented to prolong the lifespan of tebuconazole in management of ARR in apples.

Lentinan extends lifespan and increases oxidative stress resistance through DAF-16 and SKN-1 pathways in Caenorhabditis elegans.

Lentinan, extracted from Lentinus edodes, exhibits bioactive properties in vitro; however, little is known about the antioxidant potential in vivo. In this study, the effects of lentinan at 0.05, 0.25 and 1.25 mg/mL on the lifespan, locomotion, reproductive capacity, and oxidative stress resistance in Caenorhabditis elegans were determined. Compared to the untreated control, lentinan at 0.05, 0.25 and 1.25 mg/mL significantly prolonged the lifespan by 17.6%, 35.3% and 25.3% (p < 0.001), respectively, and improved the brood size, locomotion and stress resistance of the nematodes. Furthermore, lentinan at 0.25 mg/mL significantly reduced accumulation of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) by 38.1% (p = 0.013) and 49.7% (p = 0.028), respectively. In addition, lentinan at all tested concentrations significantly increased the activities of superoxide dismutase (SOD) and catalase (CAT). The expression of skn-1 and daf-16 in the treatments with lentinan at 0.25 and 1.25 mg/mL was significantly (p < 0.005) up-regulated compared with the untreated control, whereas that of the daf-2 gene was significantly down-regulated. Further evidence revealed that ROS production in lentinan-treated daf-16 and skn-1 mutant strains was similar to the untreated control. Consistent with the aforementioned results, lentinan enhanced the nuclear translocation of DAF-16 and SKN-1. Our results demonstrated that lentinan could increase lifespan and protect the nematodes from oxidative stress through DAF-16 and SKN-1.

Management of Meloidogyne incognita on Cucumber with a New Nonfumigant Nematicide Fluopimomide.

Cucumber (Cucumis sativus L.) is an economically important vegetable crop in China. Southern root-knot nematode (Meloidogyne incognita) is a significant obstacle in cucumber production, causing severe root damage and yield losses. Moreover, resistance development to fosthiazate, and poor mobility of abamectin, have led to failure to control this nematode. It is of great interest to growers and the vegetable industry to explore novel nonfumigant nematicides that can provide adequate control in an environmentally friendly manner. Fluopimomide (FM), a new chemical having a similar structure to fluopyram, was shown to exhibit toxic effects on fungi and nematodes. The efficacy of FM to reduce infection of M. incognita in cucumber was evaluated under greenhouse and field conditions. In the greenhouse, FM at all test rates resulted in a 22.5 to 39.6% and 31.3 to 55.0% reduction in the population density of M. incognita in the soil at 30 and 60 days after treatment (DAT), respectively, compared with the nontreated control. FM at 500 and 750 g ha-1 reduced (P < 0.05) root galling, meanwhile increasing plant height compared with the nontreated control at 30 and 60 DAT. In the field trials, FM at 500 and 750 g ha-1 decreased the population density of M. incognita and root galling 57.2 to 69.9% compared with the untreated control, while enhancing cucumber yield in two consecutive years. Furthermore, FM at 500 g ha-1 combined with fosthiazate was the most effective treatment showing a synergistic effect on reducing population densities of M. incognita, which was significantly greater than either FM or fosthiazate by themselves. In summary, FM has considerable potential for managing M. incognita on cucumber.

Oxidative stress, intestinal damage, and cell apoptosis: Toxicity induced by fluopyram in Caenorhabditis elegans.

Fluopyram, a succinate dehydrogenase inhibitor fungicide and nematicide, has been used extensively for agricultural pest control and toxicologically affects non-target organisms. In the present study, Caenorhabditis elegans, a well-established model organism, was used to evaluate the toxic effect of fluopyram and the possible molecular mechanisms. C. elegans was exposed to fluopyram for 24 h at three sublethal concentrations (0.01, 0.05 and 0.25 mg/L) and the physiological, biochemical, and molecular indicators were examined. The results showed that sublethal exposure to fluopyram could cause damage to growth, locomotion behavior, feeding, lifespan and reproduction of the nematodes. Fluopyram exposure induced oxidative stress as indicated by increase of ROS production, lipofuscin and lipid accumulation, and MDA level in the nematodes. In contrast, exposure to fluopyram significantly decreased the activities of target enzyme SDH and antioxidant enzymes including SOD, CAT and GST. Moreover, the expression of genes associated with oxidative stress (e.g., gst-4, sod-3, fat-7, mev-1 and daf-16), intestinal damage (e.g., mtm-6, nhx-2, opt-2, pkc-3, par-6, act-5 and egl-8), and cell apoptosis (e.g., ced-13, ced-3, egl-38, efl-2, cep-1 and lgg-1) was significantly influenced after exposure to fluopyram. According to Pearson correlation analyses, significant correlation existed between 190 pairs of parameters, which indicated that fluopyram induced multiple toxic related effects in C. elegans. These findings suggest that oxidative stress, intestinal damage, and cell apoptosis may play major roles in toxicity of fluopyram in the nematodes.

Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol.

Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; ß-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems.

Draft Genome Sequences of Pseudomonas syringae pv. tomato Strains J4 and J6, Isolated in Florida.

Pseudomonas syringae pv. tomato causes bacterial speck in tomato. We report the genome sequences of two P. syringae pv. tomato strains, J4 and J6, that are genetically closely related, with >99.9 average nucleotide identity (ANI), but vary in the presence of coronatine-associated genes.

Evaluation of a Small-Molecule Compound, N-Acetylcysteine, for the Management of Bacterial Spot of Tomato Caused by Copper-Resistant Xanthomonas perforans.

Bacterial spot caused by Xanthomonas spp. is one of the major diseases in tomato. Xanthomonas perforans is the main pathogen of bacterial spot on tomato in Florida. Currently, application of copper fungicides is the primary measure used to manage this disease. However, the development of copper resistance in X. perforans and accumulation of copper in the environment are major concerns for excessive use of copper-based products in agriculture. Due to its antibacterial properties and low environmental impact, N-acetylcysteine (NAC), a small molecule commonly used in medicine for human bacterial diseases, has been studied in agriculture for the control of plant bacterial pathogens, including X. citri and Xylella fastidiosa. This study evaluated the effect of NAC alone and in combination with copper on a copper-resistant X. perforans strain in vitro and its ability to control bacterial spot of tomato under greenhouse and field conditions. In vitro, the minimum inhibitory concentration of NAC against the X. perforans strain was 2,048 mg liter-1. NAC increased sensitivity of the copper-resistant X. perforans to copper in vitro when application of NAC was followed by copper application after 6 h. In greenhouse assays, NAC applied alone or in combination with copper significantly (P < 0.05) reduced the disease severity of bacterial spot on tomato compared with the untreated control. NAC at 100 mg liter-1 + copper at 300 mg liter-1 consistently exhibited synergistic effects against bacterial spot. In the field trials, NAC at 1,000 mg liter-1 + copper at 150 mg liter-1 significantly reduced disease severity compared with the untreated control. Results from this study demonstrated that NAC significantly reduced the disease severity of bacterial spot of tomato and enhanced the efficacy of copper against copper-resistant X. perforans, indicating that NAC could be applied for the effective management of bacterial spot of tomato.

Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus.

The fungus Magnaporthe oryzae causes devastating diseases of crops, including rice and wheat, and in various grasses. Strains from ryegrasses have highly unstable chromosome ends that undergo frequent rearrangements, and this has been associated with the presence of retrotransposons (Magnaporthe oryzae Telomeric Retrotransposons-MoTeRs) inserted in the telomeres. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, mapping, and sequencing of parental and novel telomeric restriction fragments (TRFs), along with MinION sequencing of genomic DNA allowed us to document the precise molecular alterations underlying 109 newly-formed TRFs. These included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by 'plain' telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; MoTeR-independent expansion and contraction of subtelomeric tandem repeats; and a variety of rearrangements initiated through breaks in interstitial telomere tracts that are generated during MoTeR integration. Overall, we estimate that alterations occurred in approximately sixty percent of chromosomes (one in three telomeres) analyzed. Most importantly, we describe an entirely new mechanism by which transposons can promote genomic alterations at exceptionally high frequencies, and in a manner that can promote genome evolution while minimizing collateral damage to overall chromosome architecture and function.

Prevalence and Epidemics of Neoscytalidium Stem and Fruit Canker on Pitahaya (Hylocereus spp.) in South Florida.

Since the 2000s, production of pitahaya (Hylocereus spp.) has increased significantly in South Florida. However, very limited information is available on the main diseases affecting this crop, particularly in regard to disease epidemiology and economic impact on the commodity. In this study, we surveyed five local pitahaya orchards and documented the most prevalent diseases and their causal agents. Three genera of fungal pathogens (Neoscytalidium, Alternaria, and Colletotrichum) were the major groups associated with symptoms on pitahaya cladodes (stems) during the early growing season. Among these, N. dimidiatum was identified as the most prevalent pathogen, with an overall isolation frequency of 29.8% (range, 13.9 to 47.2%). Hence, the temporal progress of N. dimidiatum stem canker infection was monitored and the relationship between stem canker intensity (incidence and severity) and fruit canker incidence was investigated. A significant positive correlation was found between fruit canker incidence and the standardized area under the disease incidence or severity curve on cladodes, suggesting that high stem canker intensity in the early season may contribute to high fruit canker incidence and thereby impact the aesthetic and market value of fruits. In vitro assays showed that both conidial germination and mycelial growth of N. dimidiatum are positively correlated with increasing temperature, with a maximum growth area at 32°C. This finding suggests a higher risk of infection, under an environment with high temperatures, which is common in South Florida. Data obtained in this study represent baseline knowledge for the future development of integrative management programs for controlling major diseases of pitahaya in South Florida.

Distribution Pattern of Thrips (Thysanoptera: Thripidae) and Tomato Chlorotic Spot Virus in South Florida Tomato Fields.

Tomato chlorotic spot virus (TCSV) is an orthotospovirus that causes a devastating disease in tomato (Lycopersicon esculentum Miller). TCSV emerged recently in South Florida. Studies were conducted in three commercial tomato fields in Miami-Dade County, Florida during the vegetable-growing seasons from October to April in 2015 through 2017. Each year, data were collected at 3, 6, and 9 wk after transplanting at various distances from the edges of each fields. Based on 3 yr total samples, three species of thrips were commonly observed melon thrips, Thrips palmi Karny (62.16 ± 0.79%), being the most abundant species followed by common blossom thrips, Frankliniella schultzei Trybom (21.55 ± 0.66%), and western flower thrips, Frankliniella occidentalis (Pergande) (16.26 ± 0.61%). Abundance of all thrips and TCSV infected plants was high at the edge of a tomato field 3 wk after transplanting with significantly fewer infected plants toward the center of the field. The distribution patterns of thrips and TCSV in various fields were mostly regular and aggregated across the sampling dates during the study period. Abundance of TCSV symptomatic plants and thrips species was high at the edge of the field and increased over time. The number of samples required to accurately determine population density of thrips was calculated by using three precision levels (0.10, 0.20, 0.30) at three predetermined densities of thrips (0.10, 0.20, and 0.40 per sample). This information will provide guidelines to growers, crop protection personnel, agricultural scouts, and researchers to develop a sustainable thrips and tospovirus management program.