Plant disease最新文献

Sweet persimmon (Diospyros kaki L.) is a fruit of significant nutritional and commercial value in Asia. In summer 2023, leaf spots were observed affecting 20 to 30% of sweet persimmon trees in a commercial orchard located in Gongcheng City, Guangxi, China. Initially, the infected leaves exhibited sparse light brown spots on their upper surface, which subsequently evolved into brown circular to irregular lesions encircled by a yellow halo. Eventually, these lesions became densely distributed across the leaves leading to insufficient nutrient accumulation in the fruit. To isolate the pathogen, diseased leaves were cut into small pieces (5×5 mm), disinfected with 75% ethanol for 15 seconds, followed by 1% NaClO for 1minute, rinsed three times with sterile water, and then transferred onto potato dextrose agar (PDA) plates. The plates were then incubated in darkness for 3 days at 25°C. Pure cultures were obtained using the hyphal-tip method and single-spore isolation. On PDA, the colonies initially appeared fluffy and white after 24 hours, turning yellowish or red after 3 days. Macroconidia (average length of 26.1 μm in length × 4.3 μm in width, n = 50) exhibited dorsiventral curvature and were hyaline, with 3 to 5 septa. Microconidia (average length of 9.45 μm in length × 3.4 μm in width, n = 50) were hyaline, aseptate, and oval. Two representative isolates, Gxfky1 and Gxfky2, were selected for further molecular analyses. Their internal transcribed spacer (ITS) region rDNA gene were amplified via PCR and sanger sequenced (GenBank Accession Nos. PP506475, PP506593) using the primer pair ITS1/ITS4 (White et al. 1990), showing more than 99% sequence identity with Fusarium kyushuense type-material strain NRRL3509 (NR_152943) according to BLASTn analysis in NCBI. To further confirm the identity of the isolates, four gene sequences were amplified: RPB1 (PP532864, PP532865), RPB2 (PP532866, PP532867), TEF1 (PP580505, PP580506), and TUB2 (PP532862, PP532863), using the F5/G2R, 5f2/11ar, EF1/EF2, and T1/T2 primer sets, respectively (O'Donnell et al., 1997; O'Donnell et al., 2010). A multi-locus maximum likelihood phylogenetic analysis revealed that Gxfky1 and Gxfky2 clustered with strains F. kyushuense with 100% bootstrap support. Pathogenicity tests using Gxfky1 and Gxfky2 were conducted on leaves of two-year-old sweet persimmon plants using non-wound inoculation. Specifically, 5-mm mycelial plugs and sterile agar plugs were placed on six leaves and secured with cling film, with six plugs each for the inoculation treatment and negative control, respectively. They were then incubated in a greenhouse at room temperature (25 ± 2°C) with a relative humidity of 70 to 80%. After 5 days, the same symptoms on naturally infected plants were observed on leaves inoculated with mycelium, while no symptoms were observed on the controls. The same fungus were reisolated from the inoculated leaves and identified based on morphology and the TEF1 gene sequence, thus ful

In the summer of 2023, the Connecticut Agricultural Experiment Station was contacted by a farm in southern Connecticut due to reports of strawberry (Fragaria × ananassa) plants showing signs of severe wilting and crown rot across multiple fields, covering ~20 hectares. Cut crowns from diseased plants had marbled red and white lesions typically associated with anthracnose crown rot (ACR). Symptomatic plants were collected from five June-bearing cultivars (cvs. AC Valley Sunset, Lyla, Dickens, and Allstar) spanning four non-adjacent fields with incidence ranging from 5-90% and severity ranging mild wilting in low incidence fields to severe wilting/mortality in high incidence fields. Internal tissue from diseased crowns was surface sterilized in 0.6% NaOCL for 3 minutes, rinsed with sterile water, and plated on potato dextrose agar. After one-week, hyphal tips of fungi were transferred to fresh plates which formed dense mycelial mats of fluffy, greyish-white hyphae. Orange spore masses formed near the center of the colonies, each of which contained numerous cylindrical and fusiform straight conidia, matching spores within the genus Colletotrichum (De Silva et al. 2019). Average conidia (n=192) length was 15.7 ± 1.6 µm and width was 5.4 ± 0.7 µm. Fungi matching this morphology were isolated from 83% of the collected symptomatic crowns and hyphae were collected from two isolates, CT5-1 and CT23-1, for DNA extraction using the GeneJET Plant Genomic DNA Purification Kit. PCR was performed using primers targeting actin (ACT), calmodulin (CAL), β-tubulin (TUB2), GAPDH (gpdA), and ITS, followed by Sanger sequencing, which yielded identical sequences for both isolates (CT5-1 Accessions numbers: PP002078-81, OR999066)(Carbone and Kohn 1999; Hassan et al. 2018; Templeton et al. 1992). These were combined with sequences from fourteen Colletotrichum genomes, all of which were aligned, trimmed, and concatenated using Mega11 (Tamura, Stecher, and Kumar 2021). Model selection was conducted using IQ-TREE and selected parameters were used to generate maximum-likelihood trees from all five loci individually and the concatenated sequence, all of which placed the isolates in a high confidence cluster with Colletotrichum siamense (Nguyen et al. 2015). To confirm the pathogenicity of the pathogen, strawberry plants (cv. Jewel) (n=5) five weeks after bare root transplant were infected. The base of each crown was penetrated 5 mm deep with a sterile 20 µL pipette tip and then inoculated with 10 µL of spores at a concentration of 106 spores/mL. Control plants (n=5) were inoculated with 10 µL of sterile water. Plants were maintained at 30°C day (16-hour)/20°C night (8-hour) in a growth chamber and assessed after 14-days. Four of the five inoculated plants had visible wilt symptoms and bisected crowns revealed the marbled red and white lesions typifying ACR. Control plants had no clear wilting and bisected cr

Verbena × hybrida, also known as common garden verbena, has an important ornamental value for their wide range of flower colors and for attracting hummingbirds and butterflies. During the winter of 2021-2022 (December through February), more than 50% pot-grown verbena plants showed symptoms of powdery mildew in a field trial at a Syngenta Crop Protection research facility in Vero Beach, FL. Symptoms were characterized by the development of white, superficial mycelium on the adaxial side of leaves which, eventually, progressed to covering the whole surface of leaves, causing leaf discoloration, shoot distortion, and eventual plant death. Morphological characterization was carried out by observing powdery mildew colonies under the microscope. This powdery mildew forms dense patches of white mycelia, mainly on the adaxial leaf surfaces. The mycelium was a mat of hyphae with septa. Conidiophores were erect. The foot cells were straight, followed by one to three short cells bearing short chains of up to four conidia. The conidia were hyaline and ellipsoidal to doliiform in shape. Conidial germination is of the Eudoidium type. The conidia ranged from 25 to 32 μm long by 12 to 16 μm wide. The length to width ratio ranged between 1.6 and 2.3, but most were between 2.0 and 2.2. This is further verification of its identity as Golovinomyces ambrosiae and not Golovinomyces latisporus, because the length to width ratio of the latter species is consistently less than 2.0 (Qiu et al. 2020). Chasmothecia were not observed. Additionally, the ITS, GAPDH, and IGS regions were sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), PMGAPDH1/PMGAPDH3R (Bradshaw et al. 2022a), and IGS-12a/NS1R (Carbone and Kohn 1999), respectively. The ITS region (GenBank number=PP924119) cannot distinguish between G. latisporus and G. ambrosiae and as such aligned 100% with both species on GenBank. However, the GAPDH and IGS regions can be used to distinguish G. ambrosiae from G. latisporus (Bradshaw et al. 2022b). The GAPDH (GenBank number=PP931995) and IGS (GenBank number=PP931996) regions aligned 100% with multiple G. ambrosiae sequences from GenBank including ON360708 and MK452567, respectively. The specimen was deposited in the Larry F. Grand Mycological Herbarium (NCSLG 24479). To confirm pathogenicity, 'Tuscany® Pink Picotee' and 'Quartz XP Violet with Eye' plugs were transplanted to 10-cm diameter pots containing ProMix potting mix and maintained in a greenhouse (± 26 °C). Inoculation was carried out 21 days after transplanting by touching infected leaves onto healthy leaves of 15 disease-free plants of each variety. Fifteen non-inoculated plants of each variety were used as controls. Typical powdery mildew symptoms and signs were first observed ten days after inoculation and the pathogen was more aggressive on 'Tuscany® Pink Picotee'. Symptoms were no

Millettia speciosa Champ, renowned for its diverse applications in traditional medicine, is extensively cultivated in the Guangxi region of China, spanning roughly 5,973 hectares. In July 2021, a plantation in Yulin, Guangxi, China (22°64'N; 110°29'E), exhibited severe leaf spot disease on M. speciosa. Notably, a 46,690 square meters area had over 40% leaf spot incidence. Initially, symptoms appeared as small, circular, pale-yellow lesions on the leaves, then turned into irregular, dark brown spots with yellow halos, leading to the wilt and defoliation of leaves. To identify the responsible pathogen, a total of five symptomatic leaves were collected and sterilized systematically. Small tissue segments (5×5 mm) from lesion peripheries were aseptically excised, then surface sterilized with 75% ethanol for 10 s, and 1% sodium hypochlorite (NaClO) for 3 min. Following this, the sterilized tissues were triple-rinsed with sterile water and cultured on potato dextrose agar (PDA) at 28 °C in the dark for 7 d. A total of seven isolates were obtained through single-spore isolation, and one representative isolate, N2-3, was selected for further analysis. After 7 d of incubation, colonies displayed flat, white, and extensively branched aerial hyphae. Over time, the reverse side of the colony changed from white to yellowish-white. The pycnidia were black with conidial droplets ranging from cream to pale yellow exuding from their ostioles. The α-conidia were one-celled, hyaline, ovoid to cylindrical, typically with one or two droplets, 2.6 to 5.9 ×1.4 to 3.9 μm (n=50). These morphological traits align with those of the genus Diaporthe, as reported by Li et al. (2022) and Crous et al. (2015). To identify the species, isolate N2-3 underwent sequencing of the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor 1 alpha (EF1-α) sections (Huang et al. 2021). Obtained sequences of ITS, BT and EF1-α (Genebank accessions nos. OR600532, OR662169 and OR662168) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP219651, ON932382, OL412437, respectively). Based on the concatenated ITS, BT and EF1-α, a neighbor-joining phylogenetic analyses using MEGA7.0 clustered with D. tulliensis. Therefore, the fungus was identified as D. tulliensis (teleomorph name) based on morphological and molecular features. A pathogenicity test was conducted on 1-year-old M. speciosa seedlings by gently abrading healthy leaves with sterilized toothpicks to create superficial wounds. Wounded leaves were then inoculated with 5 mm diameter mycelial plugs, while control seedlings received PDA plugs. Three leaves per plant and five plants per treatment were selected for assessment. All seedlings were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, the inoculated leaves showed symptoms like those in the field, while control plants remained healthy. The fungus was consistently reisolated from the in

Anthracnose caused by Colletotrichum spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of Colletotrichum spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main Colletotrichum spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (chs1) and could be performed in a few minutes (6-10 min). All isolates of C. kastii, C. fructicola and C. gloeosporioides yielded positive results using the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting DNA rapidly from tobacco leaves infected with C. kastii, C. fructicola and C. gloeosporioides. Based on these results, the RPA-LFD assay is easy to operate, rapid and equipment independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.

Plasma-activated water (PAW) exhibits potent antimicrobial properties attributed to the generation of diverse reactive oxygen and nitrogen species. This study assessed the effectiveness of PAW in vitro against phytopathogenic Xanthomonas arboricola and Pseudomonas syringae pv. syringae, which cause diseases on ornamental plants. Extending the plasma activation time of water and the incubation time of bacterial suspension in PAW increased the effectiveness of PAW. Treatments consisting of PAW activation using a power output of 200 Watts and a frequency of 50 Hz at different activation times and target population incubation times revealed significantly different effectiveness against P. syringae pv. syringae and X. arboricola. X. arboricola (reduction of 4.946 ± 0.20 log₁₀ CFU/mL) was more sensitive to PAW inactivation than P. syringae pv. syringae (reduction of 3 ± 0.15 log₁₀ CFU/mL). The plasma activation of water for 20 min followed by incubation of bacterial population for 180 min was proven to be the most effective treatment combination. The plasma activation time dose required to reduce the population by 90% was 7.47 ± 1.09 min for P. syringae pv. syringae and 4.45 ± 1.81 min for X. arboricola incubated for 180 min in PAW. The results of this study have the potential to further contribute to assessment of the effects of PAW on pathogen infected plant tissues. In addition, the findings of this study could aid in further characterization of the reactive species formed during the plasma activation of water.

Stem End Rot (SER) is a devastating post-harvest disease of mango fruits causing severe losses during storage. In 22 July 2023, 31 out of 50 intact mangoes (cv. Sensation) collected from five orchards in Huaping county (26°37'N 101°15') showed typical symptoms of SER after stored for 9 d in room temperature (24-28℃). Initially, small dark brown to black spots appeared around the fruit peduncle, which rapidly expanded through the pulp tissues. The symptomatic mangoes were surface disinfected by 3% NaClO for 30 s after soaking in 75% alcohol for 3 min, and cleaned by sterile water for 3 times. Tissues were cut from the edge of lesions, dried by sterile filter paper, transferred to PDA and cultured at 28 ℃ for 5 d (Tovar-Pedraza et al., 2020). The single-spore isolation method was used to obtain pure culture. Thirty eight isolates presented four distinct kind of morphology on PDA medium. Among them, 11 isolates with same morphology were significantly distinct from common pathogens of SER. The colonies were white and pale yellow on reverse side. Mycelia grew fast and reached the edge of 90 mm Petri dish after cultured for 5d. Pycnidia were black and scattered on the mycelial mats after 15-20 d. Conidia were fusoid, straight to slightly curved, four septa, and brown. Pigmented median cells doliiform, 14.97 - 18.62(16.11 ±0.89)×5.61- 7.28 (6.61±0.51) μm. Apical cell hyaline, subcylindrical; 1-3 tubular transparent apical appendages 12.27 - 16.68 (13.65±3.78)×1.14 - 1.99 (1.59±0.36) μm. Basal cell conical with a truncate base, hyaline, and 1-2 tubulose basal appendages with 2.85 - 7.97 (5.18±1.88)×0.99 - 1.85 (1.38±0.29) μm (n=50). These fungi were described as Pestalotiopsis kenyana. based on morphological characters (Maharachchikumbura et al., 2014) which were different from isolates characterized as other common SER pathogens (Botryosphaeria, Neofusicoccum). Based on morphology, HPSX-4 was selected for further identification. ITS region, tef1-α, β-tub of HPSX-4 were amplified and sequenced (Xun et al., 2023). The sequences were deposited in GenBank (ITS:OR889126, tef1-α:OR913431, β-tub: OR913432). The ITS, tef1-α, β-tub sequence of HPSX-4 showed 100% (525/525)，99.59% (241/242), and 100% (742/742) identity to the P. kenyana CBS442.67 sequences (ITS: NR147549，tef1-α: KM199502, β-tub: KM199395), respectively. HPSX-4 clustered with P. kenyana CBS 442.67 (type strain) based on maximum likelihood method by MEGA 7.0.21(Minh et al., 2013). Pathogenicity test was performed on 12 healthy mangoes (cv. Golek) by placing mycelial plugs around the peduncle and the middle of the fruit by pin-prick method according to Feng et al.(2023). Sterile PDA were used as control (three mangoes). Every inoculated fruit was incubated at 28°C, 95% ± 3% humidity with three replicates for each treatment. The experiment was repeated three times. Typical symptoms of SER were observed. There were no symptoms in the control group. The strain was reisolated and identified as P. k

Global change is exacerbating the prevalence of plant diseases caused by pathogenic fungi in forests worldwide. The conventional use of chemical fungicides, which is commonplace in agricultural settings, is not sanctioned for application in forest ecosystems, so novel control strategies are imperative. SIGS (Spray-Induced Gene Silencing) is a promising approach that can modulate the expression of target genes in eukaryotes in response to double-stranded RNA (dsRNA) present in the environment that triggers the RNA interference (RNAi) mechanism. SIGS exhibited notable success in reducing virulence when deployed against some crop fungal pathogens, such as Fusarium graminearum, Botrytis cinerea and Sclerotinia sclerotiorum, among others. However, there is a conspicuous dearth of studies evaluating the applicability of SIGS for managing forest pathogens. This research aimed to determine whether SIGS could be used to control Fusarium circinatum, a widely impactful forest pathogen that causes Pine Pitch Canker disease. Through a bacterial synthesis, we produced dsRNA molecules to target fungal essential genes involved to vesicle trafficking (Vps51, DCTN1, and SAC1), signal transduction (Pp2a, Sit4, Ppg1, and Tap42), and cell wall biogenesis (Chs1, Chs2, Chs3b, Gls1) metabolic pathways. We confirmed that F. circinatum is able to uptake externally applied dsRNA, triggering an inhibition of the pathogen's virulence. Furthermore, this study pioneers the demonstration that recurrent applications of dsRNAs in SIGS are more effective in protecting plants than single applications. Therefore, SIGS emerges as an effective and sustainable approach for managing plant pathogens, showcasing its efficacy in controlling a globally significant forest pathogen subject to quarantine measures.

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

Cultivation of yellow dragon fruit (Selenicereus megalanthus) in Peru has recently expanded (Verona-Ruiz et al. 2020). In August 2021, approximately 170 of 1,110 dragon fruit cuttings (15.3%) in the university's nursery (6°26'10'' S; 77°31'25'' W) showed basal rot symptoms. Initial symptoms included small brown spots on the base of stems, expanding towards the top that became soft and watery. All symptomatic plants eventually died, i.e., a severity of 100%. The disease was more prevalent on cuttings during the rooting phase than on well-established cuttings. We collected five symptomatic cuttings from throughout the nursery. Four sections of 1 × 1 cm² of tissue adjacent to the diseased area were excised from each cutting, immersed for 1 min in 2% NaClO, rinsed twice with sterile distilled water, placed on potato dextrose agar (PDA) medium (four sections per Petri plate, five plates), and incubated at 25°C for 7 days. Morphologically similar mycelia grew from all sections, and five monosporic isolates were obtained, one per plate. Colonies grew fast, reaching 60 to 64 mm in 7 days, and produced violet-white cottony aerial mycelia with orange sporodochia on PDA, and abundant macro- and microconidia on synthetic nutrient-poor agar. Macroconidia were straight to slightly curved, typically with 2 to 3 septa, 16.6 to 23.3 × 1.7 to 3.7 µm (n = 30); microconidia were oval or kidney-shaped, and commonly hyaline, 6.7 to 16.4 × 2.5 to 4.7 µm (n = 40). Genomic DNA was extracted from isolate AFHP-100, then the ITS region and the TEF1 and RPB2 partial genes were amplified and sequenced (Accession numbers PP977433, OR437358, PP537149) following Gardes and Bruns (1993) and O'Donnell et al. (1998). We conducted a BLASTn search of ITS sequence against the NCBI "nr" database and local 'megablast' searches of TEF1 and RPB2 sequences against FUSARIUM-ID v.3.0 (Torres-Cruz et al. 2022). We found 100%, 98.19 to 99.84%, and 98.81 to 99.76% identities in ITS, TEF1, and RPB2 sequences, respectively, to the ex-epitype and other reference strains of Fusarium oxysporum (CBS 144134, NRRL26406, among others). A maximum likelihood phylogenetic analysis with a TEF1-RPB2 concatenated dataset with FUSARIUM-ID sequences also showed isolate AFHP-100 was F. oxysporum. A pathogenicity test was carried out by inoculating wounded healthy roots of three cuttings with submersion in a 5 × 10⁶ conidia/ml suspension for 25 min. Then, the inoculated plants were planted in sterile soil. One cutting with wounded roots submerged in sterile water served as a control. In parallel, sterile soil was inoculated with 20 mL of the conidial suspension, and another three healthy cuttings were planted. A cutting planted in noninoculated soil also served as a control. Basal rot symptoms developed in all inoculated plants after 25 days. After re-isolation, the same fungus, corroborated based on micromorp