Phytopathology最新文献

The wheat leaf spot complex is a globally pervasive foliar disease caused by multiple fungal pathogens: Pyrenophora tritici-repentis (tan spot), Parastagonospora nodorum and Parastagonospora pseudonodorum (septoria nodorum blotch), Zymoseptoria tritici (septoria tritici blotch), and Bipolaris sorokiniana (spot blotch). Diagnostic challenges arise from overlapping symptoms and similar morphologies. We evaluated previously released molecular diagnostic tools and found that they either lacked specificity or failed to detect all species in the complex. Existing methods target different multicopy genomic regions and lacks validation against other wheat-associated pathogens. To overcome these limitations, we developed a detection tool targeting a single copy, conserved gene (β-tubulin 1, tub1) across all species. Species-specific primers were designed for multiplex PCR (mPCR) and TaqMan-based real-time quantitative PCR (qPCR), enabling sensitive, specific, and simultaneous quantification. The qPCR accurately quantified pathogen biomass with detection limits down to 0.04 pg of fungal DNA. We further showed that assays were highly accurate and species-specific when tested on wheat tissues inoculated under controlled conditions with defined single-species or mixed infections, as well as on naturally infected samples. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with selected restriction enzymes further distinguished species with unique cleavage patterns, providing an easy to use and clear identification system. Moreover, we confirmed that the assays do not cross detect barley-associated species such as the barley leaf spot pathogen Pyrenophora teres, ensuring robustness for use where host overlaps occurs. This comprehensive diagnostic provides a rapid and reliable detection, quantification of these pathogens, supporting improved disease diagnosis and enhance breeding for resistance.

Plant viruses that cause minimal to no disease symptoms may not support readily detectable virus levels. Such viruses are of concern when they persist in plant germplasm collections or in breeding populations because they can provide inoculum that can be spread and potentially cause outbreaks in susceptible plant species. The mealybug-transmitted cacao mild mosaic virus (CaMMV) causes symptomatic and asymptomatic infection of cacao trees that vary seasonally. The virus accumulates to low levels in leaves and petioles in leaves of at least some cacao genetic groups, which has confounded reliable CaMMV detection. Here, a multiplex recombinase polymerase amplification (RPA) assay was developed to increase the reliability of CaMMV detection. Three RPA primers were designed to amplify two regions of the movement protein gene (mp) of CaMMV, yielding fragments of 362 and 284 base pairs (bp). To increase detection sensitivity and specificity of CaMMV, two guide RNAs (20 bp) targeting both the CaMMV RPA amplicons were designed to activate Cas12a-mediated collateral cleavage of a fluorescent reporter. An RPA detection efficiency of 100% was achieved with respect to six known CaMMV mp variants, while the analytical sensitivity ranged from ~3 to 40 detectable CaMMV genomes. No signal was observed when cloned cacao-infecting badnavirus sequences or virus-free cacao were used as template, indicating that this assay is highly-specific for CaMMV.

Stemphylium leaf blight (SLB), caused by the fungus Stemphylium vesicarium, is a devastating foliar disease affecting onions in New York state. Primary inoculum sources for SLB epidemics potentially include infected transplants used to establish crops and volunteer onions remaining from the previous season, but little is known about their relative contribution to the population biology of S. vesicarium. In this study, 537 S. vesicarium isolates were obtained during 2022 and 2023 from infected transplants and volunteers, as well as symptomatic main crop plants collected during the mid- and late seasons. To evaluate the relative contributions of infected transplants and volunteers to S. vesicarium populations in New York, nine simple sequence repeat markers were used to characterize the genetic diversity and structure of populations by source and year. A total of 399 multilocus genotypes (MLGs) were identified, of which 27 MLGs were shared among two or more source populations and 28 between year populations. Structure analysis showed that populations from transplants were distinct from volunteers and main crop plants collected during the mid- and late seasons with low admixture. Populations had high genotypic diversity and genetic differentiation, also suggesting a minimal contribution of infected transplants to the New York S. vesicarium populations. The dominance of MLGs from volunteers to main crop populations suggests that the elimination of volunteers should be included in integrated disease management strategies. Crop rotation and hygiene practices to remove and destroy volunteer onions after harvest may be key to reducing the primary inoculum for SLB epidemics.

Globally, tomato spotted wilt virus (TSWV) is a serious pest causing tomato spotted wilt disease (TSWD) on a wide range of host plants. Integrated pest management strategies are crucial for mitigating TSWD incidence in horticultural and row crops. To assess the impact of host on TSWV, we studied four different TSWV isolates collected from peanut (TSWV-peanut), tobacco (TSWV-tobacco), and tomato (TSWV-tomato-1 and tomato-2) crops commonly grown in Georgia, United States. All four isolates were phenotypically assessed based on disease incidence and severity and serially passaged in Nicotiana tabacum 'NC196'. In addition, genomic RNAs of TSWV were measured in the newly emerged leaves of the inoculated tobacco plants. Nucleocapsid, non-structural movement, and non-structural silencing suppressor gene sequences were analyzed from passages zero, two, and four. Our findings showed 80 to 100% disease incidence in mechanically inoculated tobacco plants by the TSWV isolates studied at 21 days postinoculation (DPI). Based on the expression of symptoms in inoculated plants, high disease severity was observed in plants inoculated with the TSWV-peanut isolate. There was no statistical difference in virus copy number in the inoculated plants at 7, 14, and 21 DPI between TSWV isolates. On successive passages, virulence decreased for all the isolates (except TSWV-tomato-2). Our study indicated the difference in virulence between TSWV isolates from peanut, tobacco, and tomato crops on N. tabacum.

Bacterial and fungal pathogens threaten alfalfa production, and although chemical control is widely used, its environmental risks highlight the need for sustainable alternatives. Plant defensins, with natural origin and broad antimicrobial activity, represent promising candidates for enhancing crop resistance. However, their roles in alfalfa remain largely unknown. Here, we conducted the first genome-wide identification and characterization of the PDF gene family in alfalfa, identifying ten members grouped into two subfamilies (PDF1 and PDF2). These genes showed diverse structures, uneven chromosomal distribution, and SA/JA-responsive cis-elements, indicating involvement in biotic stress responses. Expression profiling revealed distinct tissue-specific patterns and pathogen-related induction. Comparative synteny analysis across 93 angiosperms demonstrated strong evolutionary conservation of PDF2, while PDF1 and PDF3 exhibited lineage-specific signatures. Among all MsPDFs, MsPDF2.1 emerged as a key candidate due to high expression in disease-relevant tissues, significant differential expression between resistant and susceptible germplasms, and strong evolutionary conservation. Functional assays confirmed that MsPDF2.1 confers broad antifungal and antibacterial activity and enhances pathogen resistance in Nicotiana benthamiana. BiFC indicated that MsPDF2.1 forms homomeric oligomers, consistent with a membrane-disrupting mechanism. Structural modeling revealed a conserved defensin fold with variation mainly in loop regions. This study establishes a comprehensive framework for understanding PDF diversity and identifies MsPDF2.1 as a promising resource for improving alfalfa disease resistance.

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites among the most ‎destructive agricultural worldwide causing substantial yield losses across numerous crops. ‎Current management options are limited, and the overuse of chemical nematicides poses ‎serious risks to human health and the environmental. In this study, a bacterial isolate ‎obtained was investigated as a biocontrol agent against Meloidogyne incognita, identified as ‎Pantoea conspicua strain PC (GeneBank accession no.ON203125) based on 16S rRNA gene ‎sequencing. Exposure of second-stage juveniles (J2s) caused mortality rates of 67.9%, ‎‎94.7%, 97.7%, and 99.3% in M. incognita at 12, 24, 48, and 96 hours after exposure to a ‎‎100% concentration of bacterial filtrate, respectively. In comparison, bacterial pellets ‎resulted in 18.5%, 51.8%, 62.9%, and 82.9% mortality at the same time intervals. Both, the ‎bacterial filtrate and pellets significantly inhibited egg hatchability in vitro. Greenhouse ‎experiment demonstrated that tomato plant treated with the bacterial filtrate or pellets ‎exhibited marked improvements in root and shoot growth parameters. Specifically, the ‎bacterial filtrate caused reduced the number of galls, eggs, and eggmasses per gram of root ‎by 93.12%, 97.9%, and 79.9% respectively, along with a 92.7% decrease in J2s per 250 ‎grams of soil. Overall, P. conspicua strain PC demonstrated high potential as a biocontrol ‎agent, offering advantages in its mode of action, efficacy, and contribution to ongoing ‎nematode management, while also reducing environmental impact and supporting integrated ‎pest management strategies.

Papaya ringspot virus W-type (PRSV W) and cucumber mosaic virus (CMV) severely damage cucurbits worldwide. Recently, we showed that the attenuated mutant PRSV WAC protects cucurbits against severe PRSV W infection. Here, recombinants WAC-CP, WAC-CPn, and WAC-CPc, respectively carrying the whole, N-terminal half, and C-terminal half of the CMV CP reading frame, were constructed. They induced attenuated symptoms followed by recovery in horn melon (Cucumis metuliferus) plants, similar to WAC. In Chenopodium quinoa plants, all recombinants induced infection without lesions. In horn melon plants, all recombinants remained stable after seven transfers and displayed a zigzag accumulation pattern of the beneficial protective virus, similar to WAC. From three tests with 30 plants, WAC-CP provided 100% protection one month after the challenge with PRSV W-CI or CMV, showing no severe symptoms. The absence of the challenge virus was verified by local-lesion assay and RT-PCR. Additionally, 93.3% protection was observed against the mixed challenge of W-CI + CMV. Although WAC-CPn and WAC-CPc provided high degrees of protection (76.7% - 100%) against CMV or PRSV W-CI, they only delayed the development of severe symptoms after the mixed challenge. WAC-CP was further tested in muskmelon plants, where it conferred 90.0% or 93.3% protection against W-CI or CMV, respectively, and 76.7% against the mixed challenge. The protein of individual inserts was not detected, whereas small interfering RNA was detected, suggesting that the protection against CMV is mediated by RNA silencing. Thus, WAC-CP has potential for the concurrent control of PRSV W and CMV in cucurbits.

Fusarium crown rot (FCR) has become increasingly prevalent in China's Huanghuai wheat-growing region, with Fusarium pseudograminearum emerging as the predominant causal pathogen. In this study, we developed a TaqMan qPCR assay targeting the conserved FpAH1 gene to specifically detect F. pseudograminearum. The assay's specificity was verified against 12 Fusarium species and 10 other wheat pathogenic fungi, achieving an amplification efficiency of 105.3% (R² = 0.997) and a limit of detection (LOD) of 1 × 10² copies/μL. Using this assay, we monitored the monthly dynamics of F. pseudograminearum in wheat basal stems and top soil throughout two entire growing seasons (2023-2024 and 2024-2025) under three seed treatments: untreated control, tebuconazole-coated, and cyclobutrifluram-coated. Field dynamics showed that cyclobutrifluram treatment significantly suppressed F. pseudograminearum biomass in wheat plants, while tebuconazole-coated seeds exhibited no significant difference from the untreated control. Notably, F. pseudograminearum biomass surged sharply in the late growing stage across all three treatments. Top soil maintained relatively stable F. pseudograminearum biomass, comparable to the early-stage level in wheat basal stems, across all seasons. This study established a rapid and robust TaqMan qPCR assay for specific detection of F. pseudograminearum, with broad utility in related research and practice, and documented the pathogen's cross-seasonal dynamics in both wheat plants and top soil. These characterized dynamics provide a theoretical foundation for refining Fusarium crown rot (FCR) control measures, exemplifying the assay's practical value while highlighting its potential for broader applications.

Pantoea stewartii subsp. indologenes (Psi) isolates can cause disease in several Poaceae hosts, including millets and rice, and were recently known to cause foliar and bulb symptoms characteristic of center rot in onions. Cover crops such as millet and cash crops such as corn are commonly grown in the summer after onion harvest in Vidalia, Georgia, United States. However, the risk of pathogen transmission to onions in the cropping systems in which summer crops precede onion planting is largely understudied. We evaluated the survivability of Psi in corn and pearl millet residues and assessed its ability to colonize onions transplanted into the infested soil. Our microplot study showed that millet and corn residues support the transient survival of Psi. The presence of the pathogen in the soil also overlapped with the presence of onion transplants. However, despite planting onion seedlings in Psi-infested soil, no bacterial colonization was observed in their rhizosphere and foliar surfaces. Moreover, no visible symptoms of center rot were observed in onion foliage and bulbs, indicating a lesser risk of vertical transmission in the Poaceae-Allium cropping system. We further investigated genetic determinants for bacterial survival in millet residue and bare soil by creating deletion mutants of the genes responsible for exopolysaccharides, flagellar motility, quorum sensing, and pathogenicity in a Psi pathovar cepacicola strain PNA 14-12. All mutant strains persisted for at least 24 days in millet residue at high population levels, and colonies of all the strains remained detectable in bare soil until 44 days. Exopolysaccharide production played a minor role in Psi survival, whereas none of the other targeted genes contributed to bacterial persistence in millet residue or bare soil. Overall, our findings suggest that summer crop residues play an important role in the survival of Psi in fields under an onion-millet/corn cropping scheme; however, the risk of Psi transmission from millet or corn residue to onions appears minimal. Despite this observation, crop residues should be incorporated into the soil to facilitate decomposition before onion transplanting.

Insect vectors are increasingly recognized as overlooked drivers of postharvest disease spread. Ceratocystis fimbriata, the causal agent of sweetpotato black rot, can spread rapidly in postharvest environments. Previous work established that Drosophila hydei can acquire viable C. fimbriata propagules both externally and internally, identifying this fly as a potential vector in storage facilities. Here, we expand on that finding by testing whether vector density influences disease transmission and by developing molecular diagnostic assays to improve pathogen detection. Transmission assays were conducted with four fly densities (10, 30, 50, and 80 flies) exposed to inoculum sources and then transferred to clean targets (sterile agar and uninfected sweetpotatoes, with and without wounds). Transmission occurred regardless of fly density, indicating that even small populations are sufficient to spread the inoculum, although incidence was significantly higher in wounded roots. To complement these assays, we standardized qPCR assays using dual-quencher probes targeting two C. fimbriata-specific markers (T3G9 and T5G26). Pathogen DNA was detected in both flies and asymptomatic roots, with the more sensitive marker identifying latent infections that were not visible through symptoms. Together, these results demonstrate that D. hydei vectors C. fimbriata in a density-independent manner, that wounding increases the success of infection, and that qPCR diagnostics can detect transmission events that are overlooked by visual assessment. These findings provide new epidemiological insight into postharvest black rot and support integrated management strategies that combine vector suppression with molecular surveillance.