Development of Rapid Detection Methods for Fusarium oysporum f. sp. melonis in Melon Seeds.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25-25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid, Simple DNA Preparation Protocol.

We developed an insulated isothermal PCR (iiPCR) method for the efficient and rapid detection of Fusarium oxysporum (Fo), which is a fungus that infects various hosts and causes severe crop losses. The Fo iiPCR method was sensitive enough to detect up to 100 copies of standard DNA template and 10 fg of Fo genomic DNA. In addition, it could directly detect 1 pg of mycelium and 10 spores of Fo without DNA extraction. Our study compared the performance of Fo iiPCR to that of three published in planta molecular detection methods-conventional PCR, SYBR green-based real-time PCR, and hydrolysis probe-based real-time PCR-in field detection of Fo. All diseased field samples yielded positive detection results with high reproducibility when subjected to an Fo iiPCR test combined with a rapid DNA extraction protocol compared to Fo iiPCR with an automated magnetic bead-based DNA extraction protocol. Intraday and interday assays were performed to ensure the stability of this new rapid detection method. The results of detection of Fo in diseased banana pseudostem samples demonstrated that this new rapid detection method was suitable for field diagnosis of Fusarium wilt and had high F1 scores for detection (the harmonic mean of precision and recall of detection) for all asymptomatic and symptomatic Fo-infected banana samples. In addition, banana samples at four growth stages (seedling, vegetative, flowering and fruiting, and harvesting) with mild symptoms also showed positive detection results. These results indicate that this new rapid detection method is a potentially efficient procedure for on-site detection of Fo.

First report of leaf brown blight caused by Neopestalotiopsis formicarum on jabuticaba in Taiwan.

Jabuticaba (Plinia cauliflora (Mart.) Kausel) was originated from Brazil (Lorenzi 2000). The production of jabuticaba is growing globally as its value in the food and pharmaceutical industries (Benvenutti 2021). In August 2019, jabuticaba plants with symptoms of leaf blight were observed in the field at the Meinong of Kaohsiung City, Taiwan. Disease incidence was 40%. Symptoms first presented as small, water-soaked lesions on young leaves, and then dark brown lesions of 1-3 cm in diameter on mature leaves. Six symptomatic leaves were collected from 6 jabuticaba plants for verifying the causal agents. Tissues (5 × 5 mm2) were cut from the margin of symptomatic leaf. Samples were sterilized in 1% sodium hypochlorite for 60 s, rinsed with sterile distilled water three times and then placed in 1% water agar in the dark for 5 days at room temperature. Resultant fungal colonies were purified by subculturing fungal hyphal tips on potato dextrose agar in a growth chamber (28°C, 12 h photoperiod) until fungal conidia appeared. The fungi initially produced white, cottony, aerial mycelium, after which concentric black conidiomata appeared on the plates after 7 days of incubation. The 5-celled conidia were fusiform to ellipsoid, straight to slightly curved, with sizes of 24.00-44.00 µm × 6.00-13.00 µm (avg. size, 32.00 × 9.37 µm, n = 120). The apical and basal cells were hyaline, and 3 median cells were pale brown and versicolorous. Conidia had 2-3 apical appendages and a conical basal cell with a truncate base. Based on the characteristics, which were common among isolates from diseased samples, the causal pathogen was identified as Neopestalotiopsis sp. (Solarte et al. 2018). Internal transcribed spacer (ITS), translation elongation factor 1α (TEF), ß-tubulin, and large ribosomal subunit (LSU) DNA sequences were obtained from these isolates and deposited in GenBank (MN723897, ITS; MN813055, TEF; MN813054, ß-tubulin; MN860104, LSU). Sequences demonstrated high sequence identity with those of Neopestalotiopsis formicarum ex-type cultures CBS 362.72 (Maharachchikumbura et al. 2014): 99.44% for ITS (KM199358), 99.38% for TEF (KM199517), 98.86% for ß-tubulin (KM199455), and 100.00% for LSU (KM116248). The phylogenetic relationship in Neopestalotiopsis species supported the identification of our isolates as N. formicarum. Three independent 3-isolate inoculation experiments were performed to fit Koch's postulates. Surface-sterilized leaves on live plants were punctured with a needle and inoculated with 5 µL of conidial suspension (1 × 105 conidia/mL). Inoculated plants were kept in a growth chamber (25°C, 70% relative humidity) for 7 days. Control plants were inoculated with sterile distilled water and kept under the same conditions. Inoculated leaves developed brown lesions around wounds after seven days. The pathogen was re-isolated from diseased plants, following the steps used for the original procedure, with identical characteristics as the initial isolates. This is the first report of leaf brown blight caused by N. formicarum on jabuticaba in Taiwan. N. formicarum was recently considered as a new threat to jabuticaba (Gualberto et al. 2021). In addition, it has a broad host range on many tropical crops, such as guarana and banana (Gualberto et al. 2021). Neopestalotiopsis spp. have been reported to cause important economic fruit diseases (Gualberto et al. 2021). Therefore, N. formicarum may become the potential risk for fruit production of tropical crops.

First report of anthracnose caused by Colletotrichum fructicola on tea in Taiwan.

Tea (Camellia sinensis (L.) O. Kuntze) is a very popular beverage and cash crop that is widely cultivated in tropical and subtropical areas. In November 2017, diseased tea plants that exhibiting brown blight disease were observed in Guanxi Township of Hsinchu County in Taiwan. In the plantation,15% of tea trees (about 4000 plants) had an average of 20% of the leaves with at least one lesion. The symptoms began as small, water-soaked lesions on young leaves and twigs and later became larger, dark brown, necrotic lesions of 1 to 3 cm in diameter on leaves and 2 to 5 cm in length on twigs. Symptomatic leaf tissue (1 cm2) from five samples per sample) was surface sterilized with 1% NaClO (from commercial bleach, Clorox) for 1 min, washed with sterilized water 3 times, plated onto potato dextrose agar (PDA), and incubated under 12h/12h cycles of light and darkness at 25°C until sporulation to determine the causal agent. A fungus was consistently isolated from symptomatic leaf samples (80% isolation rate). The fungus initially produced white-to-gray fluffy aerial hyphae, which subsequently exhibited dark pigmentation. Acervuli and setae were absent. The conidia were hyaline, aseptate, smooth-walled, and cylindrical with obtuse to slightly rounded ends, with sizes of 12.10 to 16.02 × 3.58 to 4.91 (average 13.77 × 4.05, n = 30) µm. The majority had two rounded guttules. The appressoria were brown to dark brown, ovoid and slightly obtuse at the tip in shape, had lengths ranging from 3.59 to 10.31 µm (with an average of 7.18 µm, n = 30), and had diameters of 3.14 to 6.43 µm (with an average of 5.10 µm, n = 30). Morphological characteristics matched the descriptions of Colletotrichum fructicola (Liu et al. 2015; Fuentes-Aragón et al. 2018). The internal transcribed spacer of nuclear ribosomal DNA (ITS), actin (ACT), chitin synthase (CHS-1), and Apn2-Mat1-2 intergenic spacer and partial mating-type Mat1-2 gene (ApMAT) sequences of the isolates were obtained to confirm this identification. The sequences showed close identity with those of C. fructicola ex-type cultures ICMP18581 and CBS 130416 (Weir et al. 2012) of 99.65% for the ITS (JX010165), 99.29% for the ACT (JX009501), and 100.00% for the CHS-1 (JX009866), as well as close identity with the other ex-type culture LF506 (Liu et al. 2015) of 99.59% for the ApMat (KJ954567), supporting the isolate's identification as C. fructicola. The sequences were deposited in GenBank, with the following accession Nos.: MN608177 (ITS), MN393175 (ACT), MT087546 (CHS-1), and MT087542 (ApMAT). Based on morphology and DNA sequence analysis, the associated fungus was identified as C. fructicola. Pathogenicity tests were performed next according to the procedures described in Chen et al. (2017). Healthy leaves on tea plants (Ca. sinensis 'Chin-shin Oolong') were wounded by pinpricking in the middle of each counterpart and inoculated with conidial suspension (1 × 107 conidia/ml, 10 µl). Both non-wounded and wounded healthy leaves were inoculated with the conidial suspension and sterile distilled water (a water control). The tea plants were covered with plastic bags to maintain high relative humidity for two days. One week after inoculation, anthracnose was observed on 40% of inoculated leaves, whereas all the control leaves remained healthy. The fungus was re-isolated from the diseased plants, and identified as C. fructicola by resequencing of the four genes. To the best of our knowledge, this is the first report of anthracnose caused by C. fructicola on tea in Taiwan although the pathogen has been present in China and Indonesia (Wang et al. 2016; Shi et al. 2017; Farr and Rossman, 2020).

Molecular Detection Assays for Rapid Field-Detection of Rice Sheath Blight.

Rhizoctonia solani (Rs), a soil-borne fungal pathogen, can result in rice sheath blight (ShB), which causes yield loss. To prevent outbreaks of ShB and enhance the sustainability of rice production, it is critical to develop a rapid ShB detection method for specific, fast, and on-site disease management. In this study, a reagent for the rapid extraction of this pathogen was developed for on-site detection. The specificity and sensitivity of a novel SMS RS1-F/SMS RS1-R primer set and a ITS1/GMRS-3 reference primer set were tested, while four different extraction protocols for ShB were developed. Moreover, intraday and interday assays were performed to evaluate the reproducibility of the detection methods developed. The results indicated that all of the developed protocols are suitable for use in detecting ShB. In addition, all the samples of infected rice yielded positive Rs detection results when subjected to TaqMan probe-based real-time PCR and SYBR green-based real-time PCR (SMS RS1-F/SMS RS1-R) tests in which automatic magnetic bead-based DNA extraction was performed. These results indicated that the two molecular detection protocols were suitable for the field diagnosis of ShB for all asymptomatic and symptomatic rice samples.

Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method.