Vascular Discoloration Research Articles

Fusarium oxysporum f. sp. apii Race 4 Threatening Celery Production in South Florida.

Fusarium wilt, caused by Fusarium oxysporum f. sp. apii (Foa), is a vascular disease affecting celery. This soilborne pathogen is classified into four distinct pathogenic races: 1, 2, 3, and 4. Notably, race 4 emerges as the most virulent, representing the latest evolutionary development of this pathogen, which was first reported in 2013 in California. In 2022, celery plants in South Florida exhibited typical Fusarium wilt symptoms, with the disease reaching a 100% incidence and causing yield losses ranging from 20 to 100%. Given the significance of celery as a vegetable crop and the severity of this outbreak, the primary objective of this study was to identify and characterize the causal agent of Fusarium wilt in South Florida. The second goal aimed to test the pathogenicity and virulence of the Fusarium isolates from Florida on celery and parsley plants. Using race-specific primers and dual-loci phylogenetic analyses, the isolates surveyed in this study were identified as Foa race 4. Pathogenicity assays in the greenhouse showed that the Foa race 4 isolate from celery induced disease not only on the two celery cultivars (Duda 30 and Duda 71) but also on two commonly cultivated parsley varieties (curly and Italian). Our study also revealed that the Foa race 4 significantly (P < 0.05) affected plant health attributes in all cultivars, including plant height, total plant weight, and root weight. Interestingly, the pathogen exhibited higher (P < 0.0001) virulence on parsley than celery based on vascular discoloration. These findings strongly indicate the urgency of comprehending and managing Fusarium wilt on celery and related crops. Furthermore, the ability of Foa race 4 to affect different plant species highlights a potential threat to agricultural production, emphasizing the need for proactive measures to mitigate the impact of this virulent pathogen.

First Report of Gibellulopsis nigrescens Causing Yellow Wilt on Chinese Cabbage in China.

Chinese cabbage (Brassica rapa L. ssp. pekinensis), in the family Brassicaceae, is a widely planted crop in China valued for its nutritional benefits. In May 2023, wilt symptoms on Chinese cabbage (cv. 'Dongtian118') were observed in several commercial fields located in Sheqi County, (32.47ºN, 112.46ºE), Nanyang, Henan Province, China. A disease survey noted that disease incidence on plants was approximately 20% to 50% within observed fields. Symptoms included yellowing and wilting leaves, and vascular discoloration of the stem bases. To isolate the pathogen, ten symptomatic leaves collected from different diseased cabbage in two field were cut into small pieces (5 × 5 mm), surface disinfected with 75% ethanol for 30 s, then washed three times in sterile water. After drying, tissues were transferred onto potato dextrose agar (PDA). Plates were incubated at 28℃ for 7 days in the dark. Twelve morphologically similar fungal isolates were obtained by single-spore subculture. The mycelia on PDA were originally white, later becoming dark gray due to the formation of masses of melanized chlamydospores after 15 days of culture. Conidiophores were hyaline and most had secondary branches. In addition, verticillate branches had three to four phialides in each whorl. The conidia were hyaline, elliptical or nearly circular, measuring from 3.2 to 9.5 × 2.6 to 3.8 μm (n=40). These morphological characteristics were similar to those described for Gibellulopsis nigrescens (Zare et al. 2007). The isolates were further identified based on PCR amplification. The ITS, GAPDH, and TEF1 genes were amplified using primers ITS1/ITS4, VGPDf2/VGPDr (Inderbitzin et al. 2011) and EF-2/EF1-728F (O'Donnell et al. 1998). BLAST analysis revealed 12 isolates were highly similar to G. nigrescens, with 99.82% similarity for ITS (OR818474, KJ534578), 93.17% similarity for GAPDH (JN188192.1, JN188166.1) and 91.07% similarity for TEF1 (EF543798.1, EF543804.1). Sequences of the representative isolate BC230515 were deposited into NCBI GenBank with accession nos. OR889646 for ITS and PP135039 for GAPDH. Pathogenicity of all 12 isolates was tested on potted Chinese cabbage plants (cv. 'Dongtian118'). Twenty-four healthy Chinese cabbage plants were inoculated by applying a 10 mL conidial suspension (1×107 conidial/mL) at the artificially wounded root region of each plant. Twenty-four control plants wounded similarly were treated with sterile distilled water. All plants were kept in a growth chamber at 22~25°C (day)/18~20°C (night) , 85% relative humidity and a photoperiod of 12 h per day. After 15 days, inoculated plants exhibited wilting symptoms similar to those observed in the field, whereas control plants remained healthy. The pathogenicity test was repeated three times. The associated fungus on the artificially inoculated plants was reisolated from the symptomatic leaves, and its identity was confirmed by PCR with the primers described above. Reisolated G. nigrescens had identical morphological and molecular characteristics to the original isolates, confirming Koch's postulates. To our knowledge, this is the first report of G. nigrescens causing yellowing and wilt of Chinese cabbage in China. G. nigrescens is a destructive pathogen with multiple hosts such as beet (Zhou et al. 2017), alfalfa (Hu et al. 2011), prevention and control measures should be taken in advance.

First report of Fusarium oxysporum f. sp. lactucae causing wilt on greenhouse-grown lettuce in North Carolina.

In August 2023, butterhead lettuce (Lactuca sativa L.) presented wilting, chlorosis, and about 2 cm of reddish-tan internal discoloration in the crown from a commercial greenhouse in Orange County, North Carolina. Plant collapse beginning with the outer leaves near the soil surface was observed with 25% disease incidence. Symptomatic lettuce plants were submitted to North Carolina State University's Plant Disease and Insect Clinic. Vascular tissue from symptomatic crowns were cut into pieces, and surface-sterilized in 10% NaOCl for two minutes. The tissue was rinsed in sterile deionized water three times, blotted dry, and placed on acidified potato dextrose agar (APDA). Three isolates, each from a different symptomatic plant, were transferred to APDA and Spezieller Nährstoffarmer agar (SNA) with pieces of sterile filter paper on the surface of the SNA media and incubated for 14 days at 23°C in the dark. Each isolate produced micro and macroconidia consistent with the morphological description of Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen (Leslie and Summerell 2006). DNA was extracted from 15-day-old fungal colonies grown on APDA media using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD, U.S.A.). The intergenic spacer locus was amplified using two primer pairs, iNL11/CNSa and iCNS1/NLa, and sequences were aligned together to form a single contig (O'Donnell et al., 2009). Primers EF1/EF2 were used to amplify the elongation factor 1-alpha region (O'Donnell et al., 1998). Each isolate was deposited into GenBank with accession numbers PP216479, PP216480, PP216481, PP235836, PP235837, and PP235838. Individual isolates revealed a 100% query cover and identity match with sequences of F. oxysporum f. sp. lactucae (FOL) and >99% identity with F. oxysporum CBS 144134 type material accessions in GenBank using BLASTn. A comparison with previously described lettuce isolates showed a homologous match with FOL race 1 isolates from California (MH412701), Arizona (DQ837658), and Greece (OQ466116), and race 4 isolates from Italy (MK801787) and Spain (OP903519). Each isolate was verified as FOL using specific primers FLA0001F/FLA0001R for FOL based on sequence tagged site markers designed by Shimazu et al. (2005). To confirm Koch's postulates, fifteen 21-day-old lettuce cv. Red Tide plants were inoculated with FOL (isolate FOLNC_660). During transplanting, lettuce roots were submerged in a 1 × 105 conidia/mL suspension for five minutes, following an inoculation protocol from Schmale and Gordon (2003). The lettuce plants were placed separately in 8.9 × 8.3 cm pots containing potting soil and maintained in a greenhouse with 31°C daytime and 25°C nighttime temperature, relative humidity of 60%, and 12-hour photoperiod. After 15 days, 80% and 86.7% of infected plants exhibited wilting, chlorosis, and vascular discoloration. The fifteen control plants remained symptomless for both experimental runs. FOL was recovered from the vascular tissue of all symptomatic plants. To our knowledge, this constitutes the first report of FOL infecting lettuce in North Carolina. Fusarium wilt of lettuce has been reported in California (Hubbard and Gerik 1993), Arizona (Matheron and Koike 2003), and most recently in Florida (Murray et al., 2020). The presence of FOL in North Carolina may result in significant crop loss for commercial growers. One of the most effective management strategies is to plant lettuce cultivars that are resistant against FOL.

First Report of Calosphaeria pulchella causing Canker and Twig Dieback of Peach (Prunus persica) in California, U.S.A.

California leads the United States in peach (Prunus persica L.) production, with approximately 505,000 tons produced in 2021 and valued at $378.3 million (California Agriculture Statistics Review, 2021-2022). During the spring and summer of 2023, twig and branch dieback were observed in three peach orchards (cvs. Late Ross and Starn) in San Joaquin County, California. Wood cankers and discoloration also occurred in branches, generally initiating at pruning wounds. Approximately 8 symptomatic twigs or branches per orchard were collected to proceed with the isolation of necrotic tissues on acidified potato dextrose agar (APDA). Isolations consistently yielded colonies of the fungal pathogen Calosphaeria pulchella (Pers. : Fr.) J. Schröt. (Réblová et al. 2004; Trouillas et al. 2012). Pure cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. Colonies on APDA grew dark pink to red or purple in their center, with a white margin. Conidiogenesis was phialidic, producing round conidial masses at the tip of phialides. Conidia were produced abundantly on APDA, and were hyaline, allantoid to oblong-ellipsoidal, 4 to 5.5 (7) × 1.2 to 2.3 μm (n = 60). Two representative isolates (SJC-62 and SJC-64) were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers and the beta-tubulin (TUB2) gene region using primers Bt2a and Bt2b. Consensus sequences of the two genes for the two isolates (ITS: PP063990, PP063991; TUB2: PP068303, PP068304) were compared to reference sequences (Réblová et al. 2015; Trouillas et al. 2012) using BLAST analysis. The ITS sequences of SJC-62 and SJC-64 were 99.8 and 99.5% identical to that of C. pulchella ex-type strain CBS 115999 (NR145357) and reference strain SS07 (HM237297); the TUB2 sequences were at least 98.5% identical to that of C. pulchella CBS 115999 (KT716476). Pathogenicity tests were conducted in 2- to 3-year-old healthy branches on 7-year-old peach trees, cvs. Loadel, Late Ross and Starn using the two fungal isolates and a control treatment (1 branch per treatment and 3 branches per tree) on each of 8-tree replicates. Branches were inoculated in June 2023 following wounding with a 5 mm cork borer to remove the bark and placing an agar plug from the margin of 10-day-old colonies on APDA directly into the fresh wound. Sterile agar plugs were used as controls. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to retain moisture. The experiment was completed twice. After four months, cankers and vascular discolorations developed around the inoculation sites. Length of vascular discoloration in inoculated branches averaged 72, 75, and 79 mm, for the Loadel, Starn, and Late Ross cvs., respectively. Calosphaeria pulchella was re-isolated from inoculated branches at 80 to 100% recovery rate, thus fulfilling Koch's postulates. The average length of vascular discoloration in the control was 13.5 mm and no fungi were recovered from control branches. Calosphaeria canker caused by C. pulchella is a global disease of sweet cherry. Recently, it was reported to cause cankers in peach trees in Chile (Grinbergs et al. 2023). To our knowledge, this is the first report of C. pulchella causing cankers and twig dieback of peach trees in the United States. These findings improve our knowledge of the etiology of canker diseases affecting peach trees and is critical for the development of effective disease management strategies.

Genetic Basis of Potato Tuber Defects and Identification of Heat-Tolerant Clones.

Heat stress during the potato growing season reduces tuber marketable yield and quality. Tuber quality deterioration includes external (heat sprouts, chained tubers, knobs) and internal (vascular discoloration, hollow heart, internal heat necrosis) tuber defects, as well as a reduction in their specific gravity and increases in reducing sugars that result in suboptimal (darker) processed products (french fries and chips). Successfully cultivating potatoes under heat-stress conditions requires planting heat-tolerant varieties that can produce high yields of marketable tubers, few external and internal tuber defects, high specific gravity, and low reducing sugars (in the case of processing potatoes). Heat tolerance is a complex trait, and understanding its genetic basis will aid in developing heat-tolerant potato varieties. A panel of 217 diverse potato clones was evaluated for yield and quality attributes in Dalhart (2019 and 2020) and Springlake (2020 and 2021), Texas, and genotyped with the Infinium 22 K V3 Potato Array. A genome-wide association study was performed to identify genomic regions associated with heat-tolerance traits using the GWASpoly package. Quantitative trait loci were identified on chromosomes 1, 3, 4, 6, 8, and 11 for external defects and on chromosomes 1, 2, 3, 10, and 11 for internal defects. Yield-related quantitative trait loci were detected on chromosomes 1, 6, and 10 pertaining to the average tuber weight and tuber number per plant. Genomic-estimated breeding values were calculated using the StageWise package. Clones with low genomic-estimated breeding values for tuber defects were identified as donors of good traits to improve heat tolerance. The identified genomic regions associated with heat-tolerance attributes and the genomic-estimated breeding values will be helpful to develop new potato cultivars with enhanced heat tolerance in potatoes.

Fusarium wilt pandemic: current understanding and molecular perspectives.

Plant diseases pose a severe threat to the food security of the global human population. One such disease is Fusarium wilt, which affects many plant species and causes up to 100% yield losses. Fusarium pathogen has high variability in its genetic constitution; therefore, it has evolved into different physiological races to infect different plant species spread across the different geographical regions of the world. The pathogen mainly affects plant roots, leading to colonizing and blocking vascular bundle cells, specifically xylem vessels. This blocking results in chlorosis, vascular discoloration, leaf wilting, shortening of plant, and, in severe cases, premature plant death. Due to the soil-borne nature of the wilt pathogen, neither agronomic nor plant protection measures effectively reduce the incidence of the disease. Therefore, the most cost-effective management strategy for Fusarium wilt is developing varieties resistant to a particular race of the fungus wilt prevalent in a given region. This strategy requires understanding the pathogen, its disease cycle, and epidemiology with climate-changing scenarios. Hence, in the review, we will discuss the pathogenic aspect and genetics of the Fusarium wilt, including molecular interventions for developing climate-smart wilt tolerant/resistant varieties of crops. Overall, this review will add to our knowledge for advancing the breeding of resistance against the wilt pandemic.

Frequency of the prominent transverse nasal root vein in children.

The presence of a vascular, blue linear discoloration on the nasal root of infants and young children is a frequent incidental feature, rarely reported in the medical literature. It is related to the trajectory of the transverse nasal root vein (TNRV). To study the frequency and clinical characteristics of the vascular discoloration of the nasal root in children. A prospective study was performed to address the presence or absence of vascular discoloration of the nasal root in all children under 6 years of age attending a pediatric dermatology clinic from November 2022 to November 2023. Data on age and skin phototype (Fitzpatrick classification I-VI) were also collected. Of 701 patients examined, 345 (49.2%) presented with a vascular discoloration of the nasal root. This was present in 97 of 193 (50.3%), 127 of 261 (48.7%), and 121 of 247 (49.0%) patients for the age groups 0-1, 1-3, and 3-6 years, respectively. The presence of vascular discoloration of the nasal root was more frequent in patients with lighter Fitzpatrick skin phototypes: 49 of 69 (71.0%) phototype II, 157 of 290 (54.1%) phototype III, and 137 of 337 (40.7%) phototype IV. A vascular discoloration of the nasal root is a frequent skin feature in infants and children, persisting at least until the age of 6. It does not constitute any medical problem aside from cosmetic concern and parents can be reassured of its benign nature. We propose the medical term "prominent TNRV" to describe this condition.

Pathogenicity and Molecular Analysis of Fusarium Isolates in Nendran and Grand Naine

Fusarium wilt, a devastating disease, poses a significant global threat to the banana industry. This study is aimed to assess the host susceptibility and pathogenicity of Fusarium isolates in tissue-cultured Nendran and Grand Naine banana varieties. Eight isolates were acquired from the Department of Plant Biotechnology at Tamil Nadu Agricultural University, Coimbatore. Among the eight isolates tested, S-6, S-32G, S-56 and S-67 produced disease symptoms in both Nendran and Grand Naine varieties. Subsequently, the pathogen was reisolated from infected tissues displaying vascular discoloration. While the isolate S-6 produced 66.67% of disease incidence in both the varieties, S-32G, S-56 and S-67 produced 100% disease incidence in Nendran and Grand Naine. Molecular analysis confirmed the presence of the Fusarium oxysporum f.sp. cubense pathogen, with an amplicon observed at 260 bp. Furthermore, non-pathogenic isolates may potentially exert direct or indirect effects on pathogenic strains, offering prospects for disease control.

First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.

First Report of Verticillium Wilt caused by Verticillium dahliae on Chilli in South Korea.

Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.

First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 μm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.

First Report of Neocosmospora mori Causing Root Rot and Stem Blight of Mulberry in Nanzhang, Hubei, China.

Mulberry (Morus alba L.) has been cultivated for thousands of years in many temperate regions in East Asia and is commonly used to feed silkworms. In May 2021, 5 to 8% incidence of stem blight on 4-year-old mulberry 'Nongsang 14' was observed in several orchards in Nanzhang County, Hubei Province, China. The roots and stems showed symptoms of vascular discoloration, and the tender new shoots, surrounded by white hyphae, were detached easily. Symptomatic stem tissues (5 mm × 5 mm) were excised from the border between diseased and healthy tissues, surface sterilized in a 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 1.5 min, washed three times in sterile distilled water, then placed on potato dextrose agar (PDA, 250 g potatoes, 2% dextrose, 1.6% agar), and incubated at 25°C in darkness. Two isolates (Bq2 and Bq3) were subcultured using the single-spore method. On PDA, colonies were cottony, with whitish aerial mycelium and the daily growth rate was 4.25 to 5.50 mm/day at 25°C in darkness. On carnation leaf agar, macroconidia were fusiform with slightly curved apical cells and foot-shaped basal cells, three to five septate, measuring 47.5 to 80.3 × 3.6 to 5.6 μm (average 68.7 × 4.7 μm, n = 30). On spezieller nährstoffarmer agar, microconidia were produced in false heads on monophialides, mostly 0-septate, oval, obovoid, or reniform in shape, measuring 5.1 to 10.7 × 2.7 to 5.3 μm (average 8.5 × 3.3 μm, n = 30). Chlamydospores were 4.9 to 11.0 μm in diameter (average 6.8 μm, n = 30), round shaped, thick-walled, and produced individually or in pairs or in chains. For molecular identification, the ribosomal internal transcribed spacers (ITS), translation elongation factor 1α (EF-1α), 28S large subunit nrDNA (LSU), and calmodulin (CAM) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1H/EF2T (O'Donnell et al. 1998), LR0R/LR5 ( Vilgalys and Hester 1990; Vilgalys and Sun 1994), and CL1/CL2A (Geiser et al. 2021; Wang et al. 2011), respectively. The sequences were deposited in GenBank (OQ711943-OQ711944 for ITS, OQ722438- Q722439 for EF-1α, OQ722441-OQ722442 for CAM, and OR116152-OR116153 for LSU). A maximum-likelihood phylogenetic analysis based on multilocus sequences was conducted using MEGA7, which showed that the two isolates grouped into a clade with Neocosmospora mori (previously Fusarium solani species complex) supported by a high bootstrap value (85%), and hence, they were identified as N. mori based on morphological and molecular analyses (Brooks et al. 2022; Crous et al. 2021; Lombard et al. 2015; Zeng and Zhuang 2023). To complete Koch's postulates, three healthy 2-month-old seedlings grown in sterile peat mix were removed from pots and the roots were washed in sterile water. Each plant was inoculated by dipping wounded and unwounded roots in a spore suspension (1 × 107 conidia/ml) for 20 min, and then 10 mL of the spore suspension was poured over the roots of each seedling after transplanting. Three plants were treated with sterilized water as a control. The tested plants were then kept in a plastic box containing sterile water and incubated at 25°C in a 12 h/12 h light/dark cycle. The pathogenicity assay was repeated three times for each isolate. Root and stem blight was observed 10 days after inoculation, while the control plants were asymptomatic. Furthermore, fungi with morphological characteristics of N. mori were only reisolated from the symptomatic stems and sequences of LSU matched those of isolates Bq2 and Bq3. This pathogen has been reported previously causing stem blight on mulberry trees in Japan and South Korea (Sandoval-Denis et al. 2019), but to our knowledge, this is the first report of N. mori causing root rot and stem blight of mulberry in China. This report will facilitate the development of effective control strategies for the disease.

Botryosphaeriaceae Species Associated with Stem Canker, Shoot Blight and Dieback of Fraxinus ornus in Italy

A severe dieback of flowering ash (Fraxinus ornus L.) has been observed in north-central Italy in the last decades. Symptoms include typical sunken, light-brown cankers on the stem and branches; vascular discoloration; tip and shoot dieback; and foliage necroses. The disease was more evident at the beginning of the growing season, and more severe on young regeneration. Six Botryosphaeriaceae species were consistently isolated from symptomatic plant tissues: Botryosphaeria dothidea, Diplodia fraxini, Diplodia subglobosa, Dothiorella iberica, Dothiorella omnivora and Neofusicoccum parvum. B. dothidea and D. fraxini expressed higher aggressiveness and showed a widespread incidence, being the species most frequently associated with cankers; the other four species were less virulent and more erratic, occurring mainly on succulent branch tips and foliage. Isolates were characterized using morphological and molecular approaches (colony/conidial phenotyping and rDNA-ITS genotyping). Phylogenetic analysis provided congruent phylogenies depicting the relationships of the six taxa with the most closely related conspecifics. Pathogenicity tests on 2-year-old seedlings confirmed the higher virulence of B. dothidea and D. fraxini. Extensive, multi-year field surveys at different sites supported the hypothesis that climatic vagaries, mainly heat, water and drought stresses, impaired tree health and vigor, facilitating infection and pervasive colonization by these Botryosphaeriaceae species. Environmental stressors are thus the key factor bringing the six fungal pathogens together in a multitrophic interaction with F. ornus in a novel, lethal fashion.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 μm x 3.77-6.84 μm (average 22.90 μm x 5.20 μm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.

Multi-species endophytic Bacillus for improved control of potato soilborne and tuber-borne diseases in Tunisia: from laboratory to field conditions

BackgroundDue to the soilborne and tuber-borne nature of their causative agents, Fusarium wilt, Rhizoctonia root canker and black dot diseases are still leading to serious problems in potato production in Tunisia and worldwide. Among endophytic bacteria, the genus Bacillus is one of the most exploited microbial groups known as potent biocontrol agents against several potato diseases. In the present study, six strains belonging to five Bacillus species were screened for their abilities to inhibit root and wilt potato pathogens in vitro, in vivo and under natural conditions over three cropping seasons and to promote plant growth.ResultsBased on the dual-culture assays, the whole-cell suspensions of SV39 and SV104 (Bacillus tequilensis), SV41 (B. subtilis), SV44 (B. methylotrophicus) and SV65 (B. amyloliquefaciens subsp. plantarum) strains exhibited potent antifungal activity against important potato soilborne phytopathogens with ~ 65 to 70% inhibition rates. Significant inhibition rates were also induced by the cell-free culture filtrates, the butanolic and the chloroformic extracts depending on the target pathogens, the concentration used and the Bacillus strain tested. In pot experiment, a decrease in Rhizoctonia root canker severity, ranging from 43 to 65% compared to the inoculated and untreated control, was induced by all Bacillus spp. strain-based treatments, while SV39-, S42- and SV65-based treatments were the most effective in suppressing by 50–53 and 65–52%, black dot severity and the relative vascular discoloration extent induced by F. oxysporum f. sp. tuberosi, respectively, relative to positive control. This biocontrol potential was associated with an enhancement of potato growth parameters. Field studies indicated that soil treatment with the most of the Bacillus spp. strains had significantly controlled all the target fungal soilborne diseases and improved at the least two growth and/or production parameters depending on the strain used and the cropping seasons. SV39-, SV41-, SV44- and SV104-based treatments resulted in a significant increase in tuber yield in one cropping season.ConclusionThese Bacillus spp. strains could be used in combinations and/or introduced with other existing practices in order to provide supplemental control of target diseases and yield promotion under organic or conventional potato production systems.

First Report of Cylindrocladiella peruviana Associated with Grapevine Black Foot Disease in China.

Black foot disease is one of the Grapevine Trunk Diseases (GTDs) that occurs in many grape growing regions and causes vine decline. Black foot disease has been reported in China in 2021 (Ye et al. 2021). In May 2022, serious root rot and nearly half brown necrosis in cross section were observed in several grapevines (Vitis vinifera cv. Cabernet Sauvignon) in Xianyang County, Shaanxi Province, China, with the incidence of 2% in 0.267 hectares. Samples (12-year-old vines) of symptomatic root were collected and taken back to the laboratory. Small fragments from the margin between healthy and diseased tissue were cut into 5 mm × 5 mm pieces. The surface was sterilized using 2% NaOCl for 2 min, followed by 75% ethanol for 30 s and rinsed three times with sterilized water. After the small pieces were dried on sterilized filter paper, they were placed onto potato dextrose agar (PDA) plates with lactic acid at 25°C for 2-3 days. The pure culture was obtained by single spore isolation. In this study, two strains were associated with black foot disease. Colony characteristics were observed in 7-day PDA plates, with cotton wool mycelium and light yellow to brown colony in reverse. Conidia were cylindrical to peanut shaped, 0-1 septa and blunt round at both ends. Size of the conidia was 7.12 to 13.89 × 2.55 to 5.16 µm (average 9.82 × 3.41 µm, n=50). For molecular phylogenetic analysis, genomic DNA of the two strains (JZB3320008 and JZB3320009) was extracted. PCR amplification was performed using four phylogenetic regions (ITS, tub2, tef1 and his3) amplified with primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (O'Donnell and Cigelnik 1997; Glass and Dnoaldson 1995), EF-688F/EF-1251R (Alves et al. 2008) and CYLH3F/CYLH3R (Crous et al. 2004), respectively. Maximum likelihood (ML) inference phylogenetic tree was constructed to confirm the identity of the two strains. ML tree reveals that the two strains clustered with Cylindrocladiella peruviana with 100% bootstrap support value. The sequences of four regions were deposited in GenBank (accession numbers OQ202205-OQ202206 for ITS, OQ225938-OQ225939 for tub2, OQ225936-OQ225937 for tef1 and OQ225934-OQ225935 for his3). Based on the morphological characteristics and molecular phylogenetic analysis, the two strains were identified as C. peruviana. To confirm the pathogenicity, the rooted cuttings (cv. Marselan) in the greenhouse were inoculated by immersing the roots in the conidial suspension (106 conidia/mL; volume, 300 mL) for 30 min, while the control was immersed in sterilized water (volume, 300 mL) for the same time. Nine plants were inoculated with C. peruviana and the same number was used as the control. These grapevines were kept in the greenhouse at 25°C. After 14 days of inoculation, the aboveground of inoculated plants showed symptoms with red leaves, while the control showed no symptoms. After 34 days of inoculation, the inoculated plants showed the reduction of the number of foliage and reduced vigor. They died with brown stem base and vascular discoloration in longitudinal section, while the control showed no symptoms. Cylindrocladiella peruviana was re-isolated by the discoloration regions and the Koch's rule was verified. To our knowledge, the pathogen has been reported in Spain and California (Agustí-Brisach et al. 2012; Koike et al. 2016). This is the first report of Cylindrocladiella peruviana causing grapevine black foot disease in China. We will better diagnose and prevent the disease in the future.

First Report of Diaporthe fruit rot and stem canker of hydroponic cucumber caused by Diaporthe sp. in the United States.

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres), D. pterocarpi, D. sclerotioides, D. sojae, and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers (Cucumis sativus) 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.

First Report and Draft Genome Resource of a Unique Opportunistic Fusarium solani Pathogen Associated with Unknown Dark Galls in Sugarbeet

Fusarium disease of sugarbeet is a threat to sugarbeet production and can cause significant losses. During routine evaluation, a unique Fusarium species was solely isolated from unknown dark galls in sugarbeets from Wilkin County, MN, U.S.A. Analysis of the RPB2 gene placed this isolate in the Fusarium solani FSSC 5 clade (100.0% similarity) but uniquely separated from the potato F. solani. Sequencing of this Fusarium species by Minion Nanopore Systems resulted in a draft genome assembly of 19 contigs with a genome size of 59.3 Mb and GC content of 50.66%. A total of 18,900 protein-coding genes, 37 secondary metabolite clusters, 1,559 secreted proteins, 668 carbohydrate-active enzymes, and 404 effectors were predicted in the genome. Pathogenicity tests showed mild vascular discoloration in inoculated sugarbeets unaccompanied by external symptoms, suggesting weak pathogenic ability of this species, indicative of an opportunistic pathogen. These results extend our understanding of this unique opportunistic Fusarium solani isolated from sugarbeet. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Host-parasite Interactions between Solanum aethiopicum, Meloidogyne incognita, and Fusarium oxysporum f.sp. melongenae as Portrayed by Disease Traits and Crop Yield

Solanum aethiopicum L. cultivation is highly constrained by wilt disease induced by Fusarium oxysporum f.sp melongenae and Meloidogyne incognita. The effects of initial population densities of these pathogens on the crop were investigated to enhance knowledge of the host-parasite interactions. The 4 x 4 factorial set of treatments were laid out in the field using the randomized complete block design with three replications. Data were collected on plant vigour, vascular discoloration, fruit weight, shoot weight, root-gall index, final nematode population, disease incidence, and disease severity. The different initial population densities of Fusarium and/or Meloidogyne spp. had detrimental effects on the crop compared to the Control. The main effects of the pathogens on the crop/wilt showed the existence of cross-over interactions for all the disease parameters. The effects of the pathogens on yield (fruit weight and shoot weight) were partially directly proportional to population densities even though the effects were significantly different (P≤0.05) compared to the Control. All the disease parameters were positively correlated. Each of the pathogens was capable of causing severe damage to the crop in either single or concomitant infection.

First report of Fusarium wilt of Cavendish bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 in Venezuela.

Fusarium wilt of banana (Musa spp.), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Dita et al., 2018). A strain of Foc that affects Cavendish (AAA) bananas in the tropics, called Foc tropical race 4 (TR4; VCG 01213), is of particular concern. Foc TR4 was first detected in Malaysia and Indonesia around 1990 but was restricted to Southeast Asia and northern Australia until 2012. The fungus has since been reported from Africa, the Indian subcontinent, and the Middle East (Viljoen et al., 2020). Foc TR4 was detected in Colombia in 2019 and in Perú in 2021 (Reyes-Herrera et al., 2020). The incursions into Latin America and the Caribbean (LAC) triggered global concerns, as 75% of international export bananas are produced in the region. Banana production in Venezuela, however, is primarily intended for domestic consumption (Aular and Casares, 2011). In 2021 the country produced 533,190 metric tons of banana on an area of 35,896 ha, with an approximate yield of 14,853 kg/ha (FAOSTAT, 2023). In July 2022, severe leaf-yellowing, and wilting, along with internal vascular discoloration of the pseudostem, were noted in Cavendish banana plants cultivar 'Valery' in the states of Aragua (10°11'8″N; 67°34'51″W), Carabobo (10º14'24″N; 67º48'51″W), and Cojedes (9°37'44″N; 68°55'4″W). Necrotic strands from the pseudostems of diseased plants were collected for identification of the causal agent using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were first surface disinfected and plated onto potato dextrose agar medium. Single-spored isolates were identified as F. oxysporum based on cultural and morphological characteristics, including white colonies with purple centres, infrequent macroconidia, abundant microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from five isolates by endpoint and quantitative-PCR using four different primer sets (Li et al. 2013; Dita et al. 2010; Aguayo et al. 2017; Matthews et al. 2020). The same isolates were identified as VCG 01213 by successfully pairing nitrate non-utilizing (nit-1) mutants of the unknown strains with Nit-M testers of Foc TR4 available at Stellenbosch University (Leslie and Summerell, 2006). For pathogenicity testing, 3-month-old Cavendish banana plants cultivar 'Williams' were inoculated with isolates from Venezuela grown on sterile millet seed (Viljoen et al., 2017). Plants developed typical Fusarium wilt symptoms 60 days after inoculation, including yellowing of leaves that progressed from the older to the younger leaves, wilting, and internal discoloration of the pseudostem. Koch's postulates were fulfilled by reisolating and identifying Foc TR4 from the plants by qPCR (Matthews et al., 2020). These results provide scientific proof of the presence of Foc TR4 in Venezuela. The Venezuelan Plant Protection Organization (INSAI) has declared Foc TR4 as a newly introduced pest (January 19, 2023), and infested banana fields were placed under quarantine. Comprehensive surveys are now conducted in all production areas in Venezuela to assess the presence and impact of Foc TR4, and information campaigns were started to make farmers aware of biosecurity protocols. Collaborative initiatives and coordinated actions among all stakeholders are needed to prevent the spread of Foc TR4 to other countries in Latin America, and to develop Foc TR4-resistant bananas (Figueiredo et al. 2023).