Virulence Comparison of a Comprehensive Panel of Xylella fastidiosa Pierce's Disease Isolates from California.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, has been found in all major grape-growing regions in California, U.S.A. Large collections of X. fastidiosa isolates are available from these areas, which enable comparative studies of pathogen genetic traits and virulence. Owing to the significant resource requirements for experiments with X. fastidiosa in grapevine, however, most studies use only a single isolate to evaluate disease, and it is not clear how much variability between isolates impacts disease development in experimental or natural settings. In this study, a comprehensive panel of X. fastidiosa isolates from all California grape-growing regions was tested for virulence in susceptible grapevine and in the model host plant, tobacco. Seventy-one isolates were tested, 29 in both grapevine and tobacco. The results of this study highlight the inherent variability of inoculation experiments with X. fastidiosa, including variation in disease severity in plants inoculated with a single isolate, and variability between experimental replicates. There were limited differences in virulence between isolates that were consistent across experimental replicates, or across different host plants. This suggests that choice of isolate within the X. fastidiosa subsp. fastidiosa Pierce's disease group may not make any practical difference when testing in susceptible grape varieties, and that pathogen evolution has not significantly changed virulence of Pierce's disease isolates within California. The location of isolation also did not dictate relative disease severity. This information will inform experimental design for future studies of X. fastidiosa in grapevine and provide important context for genomic research.

Fungicide Resistance and Host Influence on Population Structure in Botrytis spp. from Specialty Crops in California.

Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B. cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

Potential of nicotinamide adenine dinucleotide (NAD) for management of root-knot nematode in tomato.

Nicotinamide adenine dinucleotide (NAD) has been shown to induce plant defense responses to different plant pathogens, including reducing northern root-knot nematode, Meloidogyne hapla, penetration and increasing plant mass in tomato. We wanted to further evaluate NAD that are effective against the more economically important species, M. incognita and whether NAD treatments of tomato seedlings in transplant trays can protect plants in the field. Different NAD concentrations (1 mM, 0.1 mM and 0.01 mM) and three application timings (pre; post; pre and post inoculation) were evaluated in growth room and greenhouse trials. The highest tested NAD concentration (1 mM) suppressed second-stage juveniles (J2) infection for all three application methods. Root gall ratings at 30 days after inoculation (DAI) were also suppressed by 1 mM NAD compared to the other two concentrations, and egg mass number was significantly suppressed for all concentrations and application timings compared to the non-treated control. The rate of 1 mM NAD for all three application timings also improved plant growth at 30 DAI. Long-term effects of 1 mM NAD (pre, pre + post, or post applications) on nematode infection, growth and yield of tomato were evaluated in two additional experiments. All NAD applications suppressed root galls after 60 days, but only the pre + post 1 mM NAD application suppressed gall severity at 105 days, as well as suppressed egg counts by 50% at 60 DAT. No significant difference in plant biomass and fruit yield after 105 days was observed among the treatments. Two field trials were conducted in spring and fall 2020 using tomato seedlings (cv. HM 1823) treated with two different NAD concentrations (1 mM and 5 mM in spring; 5 mM and 10 mM in fall) and transplanting seedlings in fumigated (chloropicrin ± 1,3-dichloropropene) and non-fumigated plastic-mulch beds. No significant impact of NAD in terms of reducing RKN severity or overall tomato growth and production was seen in fumigated beds, but in non-fumigated beds 5 mM NAD slightly increased early fruit yield in spring, and 10 mM NAD reduced root-knot soil populations in fall.

Population Genetic Analyses of Botrytis cinerea Isolates From Michigan Vineyards Using a High-Throughput Marker System Approach.

As sequencing costs continue to decrease, new tools are being developed for assessing pathogen diversity and population structure. Traditional marker types, such as microsatellites, are often more cost effective than single-nucleotide polymorphism (SNP) panels when working with small numbers of individuals, but may not allow for fine scale evaluation of low or moderate structure in populations. Botrytis cinerea is a necrotrophic plant pathogen with high genetic variability that can infect more than 200 plant species worldwide. A panel of 52 amplicons were sequenced for 82 isolates collected from four Michigan vineyards representing 2 years of collection and varying fungicide resistance. A panel of nine microsatellite markers previously described was also tested across 74 isolates from the same population. A microsatellite and SNP marker analysis of B. cinerea populations was performed to assess the genetic diversity and population structure of Michigan vineyards, and the results from both marker types were compared. Both methods were able to detect population structure associated with resistance to the individual fungicides thiabendazole and boscalid, and multiple fungicide resistance (MFR). Microsatellites were also able to differentiate population structure associated with another fungicide, fluopyram, while SNPs were able to additionally differentiate structure based on year. For both methods, AMOVA results were similar, with microsatellite results explaining a smaller portion of the variation compared with the SNP results. The SNP-based markers presented here were able to successfully differentiate population structure similar to microsatellite results. These SNP markers represent new tools to discriminate B. cinerea isolates within closely related populations using multiple targeted sequences.

Collection and DNA Detection of Dirofilaria immitis (Rhabditida Onchocercidae), Using a Novel Primer Set, in Wild-Caught Mosquitoes From Gainesville, FL.

Dirofilaria immitis, the causative agent of dog heartworm disease, is an important cause of canine morbidity and mortality, expensive to treat, and severe infections are often fatal. Much is known about the pathogen in the canine host, yet little is known on the basic ecology of the nematode in the mosquito vector. Thus, to evaluate the effectiveness of collection techniques on ability to capture dog heartworm-infected mosquitoes (Diptera Culicidae), we conducted a field study spanning 111 wk. Four methods were used: two aspirators types, sweep netting, and a CDC trap. All sites had canines present in either residential yards (n = 4) or dog kennel facilities (n = 3). Collected mosquitoes were sorted by site, trap, species, and date, then pooled into groups of up to 25 individuals. Mosquito head and thorax pools were extracted for DNA, that was screened using currently available protocols. These protocols were found unreliable; thus, we developed a novel qPCR primer and probe set. Using this method, the original samples were re-assayed and provided 494 positive pools. Approximately 10% of positive samples were confirmed by Sanger sequencing. Twenty-two mosquito species tested positive for dog heartworm DNA, including a new association with Wyeomyia mitchellii (Theobald). Although Aedes atlanticus (Dyar and Knab), Anopheles crucians Wiedemann, and Culiseta melanura (Coquillett) composed nearly 36% of the total collection, these species represented 42% of the qPCR positive pools. Infection rates within commonly collected mosquitoes ranged up to 2.5%, with more rarely collected species ranging up to 14%. The CDC trap was the most effective collection method at trapping infected mosquitoes.

Nicotinamide adenine dinucleotide induced resistance against root-knot nematode Meloidogyne hapla is based on increased tomato basal defense.

Root-knot nematodes (RKN; Meloidogyne spp.) are among the most damaging pests to tomato production in the USA and worldwide, with yield losses ranging from 25 to 100%. Host resistance conferred by the Mi gene in tomato is effective against some species of RKN (e.g. M. incognita, M. javanica, and M. arenaria); however, there are virulent species and lines including M. hapla and M. eterolobii that break Mi-mediated resistance. Plant innate immunity is another possible form of defense against pathogen attack and is known to be induced by chemical elicitors. Nicotinamide adenine dinucleotide (NAD) is one such chemical elicitor that regulates plant defense responses to multiple biotic stresses. In this study, we investigated the role of NAD in the context of induced tomato innate immunity and RKN pathogenicity in two tomato cultivars; VFN and Rutgers, with and without Mi, respectively. Single soil drench application of NAD 24 hr before nematode inoculation significantly induced defense response pathways, reduced infective-juveniles penetration, number of galls, and increased plant mass in both cultivars. Importantly, we observed no direct toxic effects of NAD on nematode viability and infectivity. The results presented here suggest that NAD induces resistance against RKN pathogenicity likely through the accumulation of tomato basal defense responses rather than the direct effect on the infective-juveniles behavior.Root-knot nematodes (RKN; Meloidogyne spp.) are among the most damaging pests to tomato production in the USA and worldwide, with yield losses ranging from 25 to 100%. Host resistance conferred by the Mi gene in tomato is effective against some species of RKN (e.g. M. incognita, M. javanica, and M. arenaria); however, there are virulent species and lines including M. hapla and M. eterolobii that break Mi-mediated resistance. Plant innate immunity is another possible form of defense against pathogen attack and is known to be induced by chemical elicitors. Nicotinamide adenine dinucleotide (NAD) is one such chemical elicitor that regulates plant defense responses to multiple biotic stresses. In this study, we investigated the role of NAD in the context of induced tomato innate immunity and RKN pathogenicity in two tomato cultivars; VFN and Rutgers, with and without Mi, respectively. Single soil drench application of NAD 24 hr before nematode inoculation significantly induced defense response pathways, reduced infective-juveniles penetration, number of galls, and increased plant mass in both cultivars. Importantly, we observed no direct toxic effects of NAD on nematode viability and infectivity. The results presented here suggest that NAD induces resistance against RKN pathogenicity likely through the accumulation of tomato basal defense responses rather than the direct effect on the infective-juveniles behavior.

Induction of ethylene inhibits development of soybean sudden death syndrome by inducing defense-related genes and reducing Fusarium virguliforme growth.

Ethylene is a gaseous hormone that regulates plant responses to biotic and abiotic stresses. To investigate the importance of ethylene in soybean resistance to Fusarium virguliforme (Fv), the causal agent of sudden death syndrome (SDS), soybean cultivars Williams 82 (SDS-susceptible) and MN1606 (SDS-resistant) were treated 24 h before and 24h after Fv inoculation with either ethephon (ethylene inducer), cobalt chloride (ethylene biosynthesis inhibitor), or 1-MCP (ethylene perception inhibitor). Inoculated plants were grown for 21 days at 24°C in the greenhouse and then evaluated for SDS severity and expression of soybean defense genes. In both cultivars, plants treated with ethephon showed lower SDS foliar severity compared to the other treatments, whereas those treated with cobalt chloride or 1-MCP showed the same or higher SDS foliar severity compared to the water-treated control. Ethephon application resulted in activation of genes involved in ethylene biosynthesis, such as ethylene synthase (ACS) and ethylene oxidase (ACO), and genes involved in soybean defense response, such as pathogenesis-related protein (PR), basic peroxidase (IPER), chalcone synthase (CHS), and defense-associated transcription factors. Cobalt chloride and 1-MCP treatments had little or no effect on the expression of these genes. In addition, ethephon had a direct inhibitory effect on in-vitro growth of Fv on PDA media. Our results suggest that ethephon application inhibits SDS development directly by slowing Fv growth and/or by inducing soybean ethylene signaling and the expression of defense related genes.