Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Coffee Fruit Rot: The Previously Unrecognized Role of Fusarium and Its Interactions with the Coffee Berry Borer (Hypothenemus hampei).

Coffee fruit rot (CFR) is a well-known disease worldwide, mainly caused by Colletotrichum spp., the most important species being C. kahawae subsp. kahawae. In Puerto Rico, Colletotrichum spp. were identified as pathogens of coffee fruits. The coffee berry borer (CBB) was shown to be a dispersal agent of these fungi, and interaction of Fusarium with Colletotrichum affecting coffee fruits was suggested. In this study, we demonstrated that Fusarium spp. also cause CFR in Puerto Rico. Fusarium spp. are part of the CBB mycobiota, and this insect is responsible for spreading the pathogens in coffee fields. We identified nine Fusarium spp. (F. nirenbergiae, F. bostrycoides, F. crassum, F. hengyangense, F. solani-melongenae, F. pseudocircinatum, F. meridionale, F. concolor, and F. lateritium) belonging to six Fusarium species complexes isolated from CBBs and from rotten coffee fruits. Pathogenicity tests showed that F. bostrycoides, F. lateritium, F. nirenbergiae, F. solani-melongenae, and F. pseudocircinatum were pathogens causing CFR on green coffee fruits. F. bostrycoides was the predominant species isolated from the CBB mycobiota and coffee fruits with symptoms of CFR, suggesting a close relationship between F. bostrycoides and the CBB. To our knowledge, this is the first report of F. bostrycoides, F. solani-melongenae, F. pseudocircinatum, and F. nirenbergiae causing CFR worldwide and the first report of F. lateritium causing CFR in Puerto Rico. Understanding the CFR disease complex and how the CBB contributes to dispersing different Fusarium spp. on coffee farms is important to implement disease management practices in Puerto Rico and in other coffee-producing countries.

First Report of Alternaria alternata Causing Postharvest Fruit Rot of Indian Jujube (Ziziphus mauritiana) in China.

Ziziphus mauritiana Lam., commonly known as Indian jujube or ber, is a popular fruit crops grown in tropical and sub-tropical regions of China. It is commonly stored at 4℃, relative humidity of about 90%, combined with waxing or sealing with film bag. In January 2023, a postharvest fruit rot was observed on Indian jujube in three markets located in Nanchang city of Jiangxi province, China, with a disease incidence of 4 to 10%. Initially, brown spots appeared on the surface or base of the fruit, which gradually expanded into irregular brown lesions. Gray-white hyphae developed in the center of the lesions, and ultimately the fruit rotted. To isolate the pathogen, small pieces (5 × 5 mm) of ten infected fruits were surface-sterilized in 75% ethanol for 15 s and then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 3 days. Eight strains with similar morphological characteristics were isolated, and one representative isolate (JXAA-1) was used for morphological and molecular characterization. The colonies on PDA were initially olive green with white margins, and later turned dark olive or black with profuse sporulation. Conidia were borne singly or in a chain, brown, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, obclavate to obpyriform, and measured 12.9 to 33.7 × 7.5 to 12.9 µm (n = 30). On the basis of morphological characteristics, the isolates were tentatively identified as Alternaria spp. (Simmons 2007). To confirm the identification, genomic DNA was extracted from the isolate JXAA-1 with the Fungi Genomic DNA Extraction Kit (Solarbio Biotech, China). The 18S nrDNA (SSU), 28S nrDNA (LSU), internal transcribed spacer of the rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene regions (OPA 10-2) were amplified and sequenced using primers NS1/NS4, LR7/LR0R, ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, Alt-for/Alt-rev, PG3/PG2b, OPA10-2L/OPA10-2R, respectively (Woudenberg et al. 2015). The obtained DNA sequences (SSU: PP190241; LSU: PP190242; ITS: PP189927; GAPDH: PP196557; TEF1: PP196558; Alt a 1: PP196559; EndoPG: PP196560; and OPA 10-2: PP196561) showed 100% homology with those of A. alternata (GenBank accession nos. MT000349 [1020/1020 bp]; KP940477 [1312/1312 bp]; MK972909 [583/583 bp]; MN615421 [593/593 bp]; MN046379 [280/280 bp]; MN304714 [490/490 bp]; MN698284 [458/458 bp] and MH975214 [701/701 bp]). A maximum likelihood phylogenetic tree was constructed by combining all sequenced loci in IQTREE web servers. The isolate JXAA-1 clustered with Alternaria alternata (CBS 121336). The fungus associated with postharvest fruit rot on Z. mauritiana was thus identified as A. alternata. To evaluate the pathogenicity, six surface sterilized fruits were wounded by a sterile scalpel and inoculated with a 10 µl drop of spore suspension (1 × 105 conidia/ml) of isolate JXAA-1. Another six fruits were inoculated with sterilized ddH2O as control and the experiment was repeated three times. All fruits were incubated at 25°C and 80% relative humidity. After 5 days, all the wounded fruit inoculated with A. alternata showed similar symptoms to those observed previously, while the control fruits remained healthy. A. alternata was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. A. alternata has previously been reported causing leaf spot and fruit rot on Chinese jujube (Ziziphus jujuba) in China (Bai et al. 2015; Li et al. 2021). But to our knowledge, this is the first report of A. alternata causing postharvest fruit rot on Indian jujube (Z. mauritiana) in China. Therefore, managers should pay more attention to postharvest fruit rot of jujube caused by A. alternata, the foam bag is put on after the membrane bag is sealed, the broken or infected fruit is picked out in time to reduce the spread of pathogenic fungus.

First Report of Alternaria linariae Causing Postharvest Fruit Rot of Pepino (Solanum muricatum) in China.

Pepino (Solanum muricatum L.) is a popular solanaceous crop that is native to South America and is commercially grown in many countries including China for its attractive, sweet and flavorful fruits. In September 2023, a postharvest fruit rot was observed at an incidence of 7% to 10% on pepino at supermarket in Nanchang, Jiangxi, China (28.69°N, 115.81°E). Symptoms on fruits initially appeared as small black spots that later enlarged and became necrotic. To isolate the pathogen, symptomatic tissues were surface-sterilized using 75% ethanol for 15 s, then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, air dried, finally placed on potato dextrose agar (PDA) plates and incubated at 25℃ for 4 days. Ten strains (about 83% isolation frequency  from symptomatic pepino fruits) with similar morphological characteristics were isolated. The colonies on PDA were initially white, gradually turning gray and eventually becoming black, and had abundant aerial mycelia. Conidia were fusiform to linetype, dark brown, measuring 50 to 100 × 10 to 28 µm (n = 30) with 5 to 10 transverse septa and 0 to 3 longitudinal septa. Based on the morphological characteristics, the pathogen was identified as Alternaria sp. (Ma et al. 2021). To further confirm species, two representative isolates (JXAL-1 and JXAL-2) were selected for molecular identification. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1) and DNA-directed RNA polymerase II core subunit (RPB2) were amplified and sequenced by using primers ITS5/ITS4, gpd1/gpd2, Alt-for/Alt-rev, EF1-728F/EF1-986R and fRPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013; Woudenberg et al. 2014), respectively. These sequences were deposited into GenBank with accession number PP231808-PP231809 (ITS), PP238480-PP238481 (GAPDH), PP238482-PP238483 (Alt a 1), PP238484-PP238485 (TEF1) and PP238486-PP238487 (RPB2). A BLASTn homology search for these nucleotides showed 100% identity to ITS (KJ718182, 525 nt/525 nt), GAPDH (KJ718026, 579 nt/579 nt), Alt a 1 (KJ718694, 472 nt/472 nt), TEF1 (KJ718530, 334 nt/334 nt) and RPB2 (KJ718355, 772 nt/772 nt) sequences of Alternaria linariae CBS 107.61. The maximum likelihood analyses were performed for the combined ITS, GAPDH, Alt a 1, TEF1 and RPB2 using the IQtree web server (Trifinopoulos et al. 2016). In the phylogenetic tree, the isolates and isolates of A. linariae clustered together with 100% bootstrap support. Therefore, the fungus was identified as A. linariae. To evaluate pathogenicity, five healthy fruits of pepino were surface-sterilized with 75% ethanol, then wounded and a 5 mm diameter agar with isolate JXAL-1 was put on the wound. Another five fruits was inoculated with sterile agar plugs as control. All treated fruits were incubated at 25 ℃ with 80% humidity , and repeated twice. Five days later, all the wounded fruits inoculated with A. linariae showed similar symptoms and A. linariae was reisolated, while the control fruits remained healthy and no pathogen was isolated, fulfilling Koch's postulates. A. linariae is known as an important pathogen causing early blight of tomato and potato(Adhikari et al. 2020). To our knowledge, this is the first report of A. linariae causing postharvest fruit rot on S. muricatum in China, which expands the natural host range of A. linariae and will be helpful to develop efficient management strategies on pepino.

First Report of Postharvest Fruit Rot Caused by Neofusicoccum parvum on Prunus salicina (Nai Plum) in China.

Nai plum (Prunus salicina var. cordata cv. Younai) is one of the most popular fruit crop in South China. In July 2023, a fruit rot of nai plum with about 5 % disease incidence was observed in a fruit market of Changsha city, Hunan Province, China. Initially, small, brown lesions appeared randomly on the fruit surface, with disease progression, the lesions gradually expanded and developed into soft rot. To isolate possible fungi from rotten fruits, small pieces (2 × 2 mm) from the periphery of 10 infected fruits were surface-sterilized using 70% ethanol for 10 s, rinsed three times in sterile distilled water, air dried, and then placed onto potato dextrose agar (PDA) plates and incubated at 28℃ for three days. Emerging colonies were subcultured by hyphal tip transfer on fresh PDA. A total of ten isolates with similar morphology were obtained. Fungal colonies were initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidia were hyaline, single celled, ellipsoidal to fusiform, and range from 12.7 to 20.0 µm long (avg. 16.9 ± 2.39 µm) × 5.3 to 7.3 µm wide (avg. 6.3 ± 0.82 µm). These morphological characteristics of these isolates matched those of Neofusicoccum parvum (Phillips et al. 2013). To future confirmation of the identify, the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-a), and beta-tubulin TUB2) genes of two representative isolates (JXNP1 and JXNP2) were amplified and sequenced using primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999; Phillips et al. 2013), and BT2A/BT2B (Glass and Donaldson 1995), respectively. The sequences of both isolates were deposited in GenBank for the ITS (accession nos. OR899331 and OR899332), TEF1-a gene (accession nos. OR909890 and OR909891) and TUB2 gene (accession nos. OR909892 and OR909893). BLAST analysis showed 99-100% identity with the ex-type strain of N. parvum (CMW9081) for ITS, TEF1-a and TUB2. A maximum likelihood phylogenetic tree was constructed using IQtree web server based on combined ITS, TEF1-a and TUB2 data set. The phylogenetic tree revealed that two isolates clustered with N. parvum in a clade with 90% bootstrap support. Based on morphological and molecular data analysis, the isolates were identified as N. parvum. To confirm the pathogenicity, five healthy nai plum fruits were wounded by using a sterile needle after surface sterilization with 75% ethanol, then a 5-mm-diameter mycelial disc of isolate JXNP1 was taped to the wound, the control fruits were taped with sterile agar plugs. All fruits were incubated at 25 ℃ with 80% humidity. After five days, typical naturally occurring fruit rot symptoms appeared on the fruits which inoculated with N. parvum, whereas control fruits remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and comfirmed as N. parvum by morphological and molecular analysis. Previous studies reported that N. parvum caused fruit rot on various common fruits in China, including loquat, kiwifruit and citrus (Lei et al. 2013; Zhai et al. 2019; Zhou et al. 2013). To our knowledge, this is the first report of N. parvum causing postharvest fruit rot on nai plum in China. This finding provides critical insights for the management of the high-risk disease on plum in China.

First report of Botryosphaeria dothidea causing fruit rot on Chinese olive (Canarium album Raeusch.) in Guangdong province of China.

Chinese olive (Canarium album Raeusch.) is a traditional Chinese medicinal plant, mainly cultivated in Guangdong and Fujian provinces in China (Lai et al. 2022). In October 2023, Chinese olive fruit spots were observed in all the Chinese olive orchards surveyed in Chaozhou city (23.75°N, 116.67°E) of Guangdong, with an incidence up to 15%. Early disease symptoms on fruits appeared as circular or irregular, dark brown to black spots with yellowish lesions, and later the spots slowly coalesced to form large necrotic areas, which seriously affected the fruit marketability. To isolate the causal agent, small pieces (~0.3 mm2) of fruit tissue were excised from the lesion margins, and surface-disinfested with 75% (v/v) ethanol for 1 min, followed by 1% NaClO for 3 min, and rinsed three times with sterile water. The pieces were then placed on potato-dextrose-agar (PDA) and incubated at 27°C. Ultimately, four fungal isolates were obtained with similar morphology phenotypes, colonies initially appeared white with irregular margins and after 4-6 days turned dark gray gradually with dense aerial myceliu. Microscopy revealed conidia were single-celled, hyaline, aseptate, fusiform to subclavate, and measured 18.1-22.5 µm × 6.4-9.3 µm (19.8 × 7.4 m on average, n = 30), which were consistent with those descriptions of Botryosphaeria dothidea (Vasic et al. 2013; Zhang et al. 2023). To further identity the isolates, partial sequences of ribosomal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS5, TEF-F/R, TUB2-F/R, respectively (Xu et al., 2023; Hong et al. 2006). The sequences of four isolates were identical, and those of representative strain GDCZ-1 were deposited in GenBank (ITS, OR584295; TEF1-α, OR685157; TUB2, OR685158). Using Neighbor-Joining algorithm, phylogenetic tree based on concatenated sequences of ITS, TEF1-α, and TUB2 showed that GDCZ-1 clustered with B. dothidea. To fulfill Koch's postulates, pathogenicity tests were performed on healthy Chinese olive fruits using the needle-prick inoculation method. The fruits were wounded with a sterile needle at the equatorial area (depth of 3-4 mm), and inoculated with 10 µL of spore suspension (106 /mL). The control fruits were inoculated with sterile water. Inoculated fruits were placed in sterile plastic containers to maintain high relative humidity (almost 100%) and incubated at 27°C. After 4 days, the inoculated fruits showed similar symptoms with those observed in the field infected fruits, while the control remained asymptomatic. Pathogen re-isolated from the inoculated fruits showed identical morphological characteristics to the original isolate GDCZ-1. As far as we know, fruit rot caused by Alternaria alternata has been recently reported on C. album in China (Shao et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. album in Guangdong. Our report will provide crucial information for studying the epidemiology and management of this disease.

First Report of Neofusicoccum parvum Causing Fruit Rot on Psidium guajava in China.

Guava (Psidium guajava L.), a nutrient-rich and economically significant fruit, is extensively cultivated in southern China. In six continuous years (from 2018 to 2023), dark-purple rotted guava with sunken lesion were observed on guava trees (variety 'Zhenzhu', aged over 5 years) in Dongguan and Panyu districts, Guangdong Province. Annually, the incidence of fruit rot disease in the affected fields reached 30% to 50% and significantly reduced the yield and quality of harvest guava. The initial symptoms on the epicarp of the fruits were black, needle-like dots that rapidly spread, causing partial or complete fruit rot within two to three weeks. To identify the causative agent, six symptomatic fruits were collected from two different orchards. Samples of 0.5 cm³ were excised from the lesion margins of each fruit. These samples were surface-sterilized with 70% ethanol for 30 seconds, followed by 0.2% NaClO for 2-3 minutes, and rinsed in sterile water three times. The samples were then cultured on potato dextrose agar (PDA) at 25°C for five days. This process yielded eight fungal isolates with similar morphological. Initially, the colonies were white with dense aerial mycelium becoming dark gray after 4 to 5 days. The mycelia were septate and branched. No spores were observed on PDA. To induce spore formation, the isolates were cultured on water agar for 20 days. This process led to the production of hyaline, aseptate, ellipsoid conidia, which were thin-walled, smooth-surfaced, and measured 3.7-5.1 × 1.6-2.2 µm (n = 100). Three isolates, including at least one from each orchard (Np1, Np2, Np3), were selected for further analysis. Genomic DNA was extracted using Axygen MAG-FRAG-I-50 (Axygen Bio-Tek). The internal transcribed spacer of rDNAs (ITS), beta-tubulin (tub2), the nuclear ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α) gene regions were amplified using the primers ITS1/4, Bt2a/Bt2b, LR5/LR0R, and EF1-728F/EF1-986R (Golzar and Burgess 2011) and sequenced. Sequence analysis using MEGA 7.0 (Kumar et al., 2018) revealed 100% similarity among the isolates. BLAST searches of the ITS, tub2, LSU, and tef1-α sequences (accession nos. MN907637, MT537938, MT528156, MT537939) showed the highest nucleotide similarities (99.24 to 100%) to Neofusicoccum parvum strains (Crous et al. 2006). A phylogenetic tree was constructed with MEGA 7.0 based on the nucleotide sequence tub2 using the maximum likelihood method. Pathogenicity tests on 10 healthy guava fruits using mycelium-inoculated and control fruits confirmed the causative agent. The inoculated fruits, maintained at 25°C under a 12-h light/dark cycle, exhibited symptoms identical to the field infections within four to seven days, while control fruits remained symptomless. The fungus, reisolated from the inoculated fruits, was morphologically identical to the original isolates, fulfilling Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, N. parvum as the causal agent of the fruit rot disease on guava. Previously, N. parvum has been reported in association with fruit rot on Eriobotrya japonica and Juglans regia (Zhai et al. 2019; Chen et al. 2019). To our knowledge, this is the first report of N. parvum affecting guava in China.

First report of brown spot on stored apple fruits caused by Stemphylium vesicarium in Serbia.

Apple is one of the most economically important fruit crops worldwide, and fungal postharvest diseases can cause significant losses during storage (Petres et al. 2020). Apple fruits (cultivar Fuji) with necrosis symptoms were collected during the fall of 2022 from the cold storage facility (ULO - Ultra Low Oxygen) in Titel, Serbia. The fruits originated from the apple orchard in Titel, Serbia (45°12'47.1"N, 20°15'23.6"E). The pathogens were isolated from collected fruit samples using standard phytopathological techniques. Fruits were surface-sterilized, rinsed with sterile water, aseptically cut in half, and small fragments collected from the border of healthy and diseased tissue were placed into Petri dishes on Potato Dextrose Agar medium (PDA) and incubated at 25±1 °C in dark for seven days. The obtained 11 isolates were identified to the genus level as Alternaria (incidence 46%), Penicillium (36%), Fusarium (9%) and Stemphylium (9%) based on morphological characteristics. Pathogenicity of all isolates was confirmed on apple fruits of cultivars Fuji and Golden Delicious. The fruits were surface-sterilized, sprayed with 5 ml conidial suspension (1×105 conidia/ml) and incubated at room temperature for 21 days. Symptoms developed on inoculated fruits were the same as symptoms observed on apple fruit samples collected from cold storage. Reisolation from artificially inoculated fruits resulted in colonies that morphologically corresponded with the colonies used for inoculation. Stemphylium isolate was the only one included in further research. Initial symptoms and symptoms on artificially inoculated apple fruits caused by Stemphylium sp. occurred as circular dark brown necrosis located near the calyx, without visible sporulation on the fruit surface. The isolate and reisolate formed aerial, white to light brown mycelia. The pigmentation of the culture medium was pale to dark brown. Conidia were singular, cylindrical and multicellular, brown to dark brown, 22-35.1 long and 12.6-18.9 µm wide. Based on morphological properties, isolate and reisolate were identified as Stemphylium vesicarium which is in line with the description reported by Sharifi et al. (2021) and Gilardi et al. (2022). The identification of S. vesicarium isolate was confirmed by polymerase chain reaction (PCR) by amplifying and sequencing three regions using following primer pairs: Bt2a (5'- GGT AAC CAA ATC GGT GCT GCT TTC -3') and Bt2b (5'-ACC CTC AGT GTA GTG ACC CTT GGC-3') for ß-tubulin region (Nasri et al. 2015), ITS1 (5'-TCC GTA GGT GAA CCT GCG G - 3') and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC-3') for ITS region (White et al. 1990), and EF1 (5' - ATG GGT AAG GAG GAC AAG AC - 3') and EF2 (5'- GGA AGT ACC AGT GAT CAT GTT - 3') for TEF-1α region (O'Donnell et al. 1998). PCR products were separated by horizontal gel electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and visualization under UV light revealed amplified fragments of the expected size of 500 bp for Bt2a/ Bt2b primer pair, 600 bp for ITS1/ITS4 primer pair, and 700 bp for EF1/EF2 primer pair. The obtained amplicons were Sanger sequenced (Macrogen Europe BV) in both directions. BLASTn analysis showed the identity of amplified fragments of the isolates with sequences of S. vesicarium present in the GenBank of 100% (MT881940.1 and JQ671944.1) for the ß-tubulin region, 99.40% (MT520589.1 and OR256793.1) for the ITS region, and 99.49% (DQ471090.2 and MT394642.1) for the TEF-1α region. The sequences were deposited to NCBI GenBank (Accession No. OQ653540 for the ß-tubulin region, OQ678016 for the ITS region, and OR232710 for the TEF-1α region). To our knowledge, this is the first finding of S. vesicarium on apple fruits in the Republic of Serbia, and the finding of a new causal agent of postharvest apple fruit rot.

Utilizing Disease Prediction Models to Time Fungicide Applications for Controlling Ripe Rot, Caused by Colletotrichum spp., in Maryland Vineyards.

Two previously published ripe rot prediction models, DF2-NN and GH2-DT, were evaluated for fungicide application timing efficacy in Maryland vineyards. Both models utilize leaf wetness duration (LWD), temperature, and grape cluster phenological stages as model parameters. These three parameters were tracked throughout the 2021 to 2023 seasons in three vineyards. The fungicide efficacy trials started at the veraison phenological stage and included a nontreated control, a 12-day interval treatment, and two model-triggered treatments when risk predicted by the models crossed a threshold. The severity of ripe rot on the clusters in each treatment was assessed when the fruit were mature. Ripe rot severity in the nontreated controls was higher during seasons with more LWD and more precipitation. Days in which the models were triggered by the environmental conditions primarily coincided with precipitation events and lengthy LWDs. The model-triggered treatments never had significantly higher ripe rot severity than the 12-day interval treatment but had significantly lower severities than the nontreated control in most trials which had high ripe rot pressure. Furthermore, the model-triggered treatments resulted in fewer fungicide applications than the 12-day interval treatment on average. The DF2-NN model appeared to be more accurate and useful for ripe rot prediction and treatment than the GH2-DT model because it triggered fewer fungicide applications while reducing ripe rot. This model may be useful for improving or maintaining ripe rot control with fewer fungicide inputs, which may be beneficial for the environment and the reduction of fungicide resistance selection.

First Report of Botryosphaeria dothidea Causing Postharvest Fruit Rot of Plum in China.

The Nai plum (Prunus salicina var. cordata cv. Younai) holds significance as an important deciduous fruit crop in China. In July 2023, symptoms of postharvest fruit rot were observed on Nai plum with a 10% disease incidence of harvested fruits in three supermarkets, located in Nanchang City, Jiangxi Province, China. Infected fruits displayed brown, circular lesions, accompanied by a transition in the surrounding peel color from cyan to red. To investigate the causal agent, small sections (3 to 4 mm2) from the periphery of ten infected fruits were subjected to surface sterilization using 75% ethanol for 30 seconds. Following sterilization, the samples were rinsed three times with sterilized distilled water, air-dried, and aseptically placed on potato dextrose agar (PDA) at 25 ℃ for 3 days. Isolated colonies were subcultured by hyphal tip transfer. Ten of the resulting 12 fungal isolates showed similar morphological characteristics. The colonies exhibited an initial white hue, gradually transitioning to gray, and featured short and thick aerial hyphae with an irregular colony margin. Microscopic examination revealed conidiogenous cells that were hyaline, aseptate, and narrowly fusiform. The conidia were measured 11.0 to 15.6 × 3.2 to 4.9 µm (x̅ = 13.5 ± 1.4 × 4.0 ± 0.4 µm, n = 30), and were hyaline and subcylindrical. The morphological characteristics were in accordance with those of the Botryosphaeria species (Crous et al. 2006). To identify the strain, two representative isolates, JFRL03-1792 and JFRL03-1793, were selected for further characterization. Amplification of nucleotide sequences from three regions (ITS, TEF1-a and TUB2) was conducted using the primer sets ITS5/ITS4, EF1-728F/EF1-986R, and BT2A/BT2B, respectively (Guo et al. 2023). The resulting sequences were deposited in GenBank under the accession numbers: OR418373 and OR418374 for ITS; OR424405 and OR424405 for TEF1-a; OR424411 and OR424412 for TUB2. A BLASTN homology search of the obtained sequences revealed a high similarity of 99%-100% to the ITS (AY236949, 511/513 nucleotides), TEF1-a (AY236898, 282/282 nucleotides), and TUB2 (AY236927, 431/431 nucleotides) sequences of Botryosphaeria dothidea CWM8000 (ex-type). Maximum likelihood analyses were performed for the combined ITS, TEF1-a, and TUB2 dataset using Phylosuite V1.2.2 (Zhang et al. 2020). The resulting phylogenetic tree indicated that the two representative isolates were clustered together with Botryosphaeria dothidea in a clade with 95% bootstrap support. Based on the comprehensive assessment of morphological and molecular data, the two isolates were conclusively identified as B. dothidea. To confirm pathogenicity, six healthy Nai plum fruits were surface sterilized with 75% ethanol and were subsequently wounded with a sterile needle. A 5-mm-diameter mycelial plug of the isolate JFRL03-1792, cultured on PDA at 25 ℃ for three days, was applied to the wound. Another set of six fruits was inoculated with sterile agar plugs as control. Following incubation in a climatic chamber at 25 ℃ and 80% relative humidity, the fruits were examined after 5 days. The experiment was repeated twice. The fruits inoculated with B. dothidea displayed typical rot symptoms, while the control fruits remained asymptomatic. In adherence to Koch's postulates, the fungus was successfully re-isolated from the inoculated fruits and confirmed as B. dothidea through morphological and molecular analysis. B. dothidea has previously been reported causing fruit rot on kiwifruit, winter jujube, and apple (Tang et al. 2012; Zhou et al. 2015; Marsberg et al. 2017; Xu et al. 2023). In addition, B. dothidea also reported causing Botryosphaeria canker disease on plum (Lin et al. 1994). But to our knowledge, this is the first documentation of B. dothidea causing postharvest fruit rot on plum in China. This discovery imparts critical insights into the management of this high-risk disease affecting plum in China.

A Semi-Selective Medium to Evaluate Over-Summering Survival of Neopestalotiopsis sp. in Florida Strawberry Fields.

Devastating disease outbreaks with leaf spot symptoms and fruit rot caused by a fungus identified as a cryptic species of Neopestalotiopsis have generated concern in the Florida strawberry industry. Some Neopestalotiopsis species are saprobes in soil and plant debris, but the ability of the new Neopestalotiopsis sp. to survive in strawberry debris was unknown. Therefore, the objectives of this study were to develop a semiselective medium for isolation of Neopestalotiopsis spp. and to evaluate Neopestalotiopsis sp. survival in soil and strawberry plant debris over the summer in Florida. The Neopestalotiopsis semiselective medium (NSM) inhibited growth of most fungal species pathogenic to strawberry in Florida, except Neopestalotiopsis rosae, Neopestalotiopsis sp., and Phomopsis obscurans, in addition to Penicillium spp., which are nonpathogenic. However, Neopestalotiopsis species, P. obscurans, and Penicillium spp. could be distinguished in the medium through morphological characteristics. Soil samples arbitrarily collected from six commercial fields toward the end of the season (May) and before (July) and after (September) preplant soil fumigation in the following season were processed with NSM, and Neopestalotiopsis spp. populations were calculated as CFU per gram of soil. CFU ranged from 48.3 to 2,410.8 at the end of the season and from 1.7 to 630.8 before soil fumigation, but Neopestalotiopsis spp. were not recovered after soil treatment. However, 1.7 to 25 CFU were obtained from the nontreated areas in the row middles. Neopestalotiopsis sp. survival was also evaluated on diseased strawberry plants (leaves and crowns) exposed to the environment for 17 months. On leaves, Neopestalotiopsis spp. were recovered for 6 months before leaf decomposition, whereas the number of CFU on crowns declined monthly, but colonies were still recovered during the final evaluation months. A high-resolution melting analysis confirmed most of the colonies from soil and crowns were the new Neopestalotiopsis sp. Our results show Neopestalotiopsis sp. can survive in soil and strawberry debris under Florida summer conditions and may serve as a source of inoculum for the subsequent season.

First report of Erwinia pyrifoliae causing flower blight and fruit rot on greenhouse-grown Fragaria × ananassa in the United States.

Erwinia pyrifoliae causes disease of pear (Pyrus spp.), apple (Malus spp.), and strawberry (Fragaria × ananassa) (Wenneker and Bergsma-Vlami 2015), which are economically important commodities in the US. Disease symptoms on pear and apple are indistinguishable from those caused by the non-quarantine fire blight pathogen, E. amylovora (Kim et al. 1999), which also causes disease on strawberries (Atanasova et al. 2005). Samples of greenhouse-grown strawberry 'Albion' from Ohio were submitted to the Purdue Plant and Pest Diagnostic Lab in December 2023. Fruits were stunted with brown lesions, while sepals and pedicels had brown-black water-soaked lesions. Cut fruit exuded bacterial ooze from the main vascular bundle. Bacterial streaming was observed microscopically from symptomatic tissue which tested positive with the E. amylovora ImmunoStrip® (Agdia Inc., Elkhart, IN); reported by the manufacturer to cross-react with E. pyrifoliae. Isolation from symptomatic tissue produced pure cultures on sucrose peptone agar and Kings medium B after incubation at 27°C for 48 hr, and colonies appeared circular and white/opaque. Crude DNA extractions were prepared by boiling colony suspensions in Tris-EDTA buffer. Two independent real-time PCR tests specific for E. pyrifoliae (Lehman et al. 2008; Yasuhara-Bell et al. 2024) produced positive results. Conventional PCR using an E. pyrifoliae-specific primer set targeting a divergent region between pstS and glmS genes (Wensing et al. 2011) also produced positive results. The amplicon was Sanger-sequenced and deposited into NCBI GenBank (Accession PP757383). BLASTn analysis using the Nucleotide collection and Whole-genome shotgun contigs revealed top matches (100% query coverage; 97.5% identity to type strain DSM 12163) with E. pyrifoliae only; next closest match was E. amylovora (53% query coverage). To confirm Koch's postulates, immature fruit of six healthy strawberry 'Albion' plants were wounded with a sterile pipette tip and then submersed in a bacterial suspension in sterile deionized water (DI H2O) (3.1×107 cells/ml). Fruit of six additional plants were mock inoculated using sterile DI H2O. Plants were placed in plastic bags for 48 hr at room temperature with a 12-hr photoperiod. Symptoms were first observed on inoculated plants 1.5 days post-inoculation (DPI). Brown discoloration was observed within fruit and as spreading lesions on fruit pedicels by 4 DPI; mock-inoculated plants remained asymptomatic. Bacterial streaming from symptomatic tissue allowed successful re-isolation of the bacterium. Molecular testing confirmed isolates to be E. pyrifoliae, thus completing Koch's postulates. Following initial confirmation, additional samples of infected strawberry ('Albion' and 'GB96') from the same greenhouse were confirmed positive for E. pyrifoliae by molecular testing and sequencing. To our knowledge this is the first time Erwinia pyrifoliae was detected in the US. There are many known pathways of introduction from Asia and Europe; however, pstS-glmS sequence comparison with strawberry isolates from the Netherlands (sequences provided by M.J.C. Pel) suggests this US strawberry strain is unique, but most closely related to Japanese strains (98.5% identity). Potential origin of this strain is unknown, but comparative genomics studies to investigate relatedness among strains are planned.

Diversity and characteristics of Fusarium solani species complex (FSSC) isolates causing collar rot and fruit rot of passion fruit in Taiwan.

Fusarium solani species complex (FSSC) is a causal agent of collar rot and fruit rot in passion fruit worldwide. This study investigated the diversity and characteristics of FSSC isolates causing collar rot and fruit rot in Taiwanese passion fruit. Thirty-five FSSC isolates were harvested from collar rot and fruit rot samples of passion fruit from various cultivars and different geographical locations in Taiwan. The majority of these FSSC isolates caused collar rot and fruit rot disease of varying virulence in the stems and fruits of the purple and yellow cultivars of passion fruit. FSSC isolates were categorized into four groups: F. solani-melongenae (FSSC 21; n=29), F. solani (FSSC 5; n=1), F. liriodendri (FSSC 24; n=1), and an unknown group (n=4) based on the phylogenetic analysis of internal transcribed sequence (ITS), translation elongation factor 1 alpha (TEF-1α), and RNA polymerase II subunit 2 (RPB2) sequences. In Taiwan, F. solani-melongenae was the dominant species causing collar rot and fruit rot in passion fruit. F. solani-melongenae was a homothallic fungus that produced perithecia in diseased tissues. However, F. solani and F. liriodendri did not produce perithecia. The unknown FSSC group showed morphological characteristics similar to F. solani-melongenae and produced perithecia. Phylogenetic analysis based on the ITS and TEF-1α sequences demonstrated that the Taiwanese FSSC isolates were distinct from the Brazilian and Chinese FSSC isolates. In summary, FSSC isolates causing collar rot and fruit rot of Taiwanese passion fruit showed high diversity, potentially associated with the geographical locations.

Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States.

The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [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.

The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.).

BACKGROUND: The cultivated strawberry (Fragaria × ananassa Duch.) is one of the most economically important horticultural crops worldwide. Botrytis fruit rot (BFR) caused by the necrotrophic fungal pathogen Botrytis cinerea is the most devasting disease of cultivated strawberries. Most commercially grown strawberry varieties are susceptible to BFR, and controlling BFR relies on repeated applications of various fungicides. Despite extensive efforts, breeding for BFR resistance has been unsuccessful, primarily due to lack of information regarding the mechanisms of disease resistance and genetic resources available in strawberry. RESULTS: Using a reverse genetics approach, we identified candidate genes associated with BFR resistance and screened Arabidopsis mutants using strawberry isolates of B. cinerea. Among the five Arabidopsis T-DNA knockout lines tested, the mutant line with AtWRKY53 showed the greatest reduction in disease symptoms of BFR against the pathogen. Two genes, FaWRKY29 and FaWRKY64, were identified as orthologs in the latest octoploid strawberry genome, 'Florida Brilliance'. We performed RNAi-mediated transient assay and found that the disease frequencies were significantly decreased in both FaWRKY29- and FaWRKY64-RNAi fruits of the strawberry cultivar, 'Florida Brilliance'. Furthermore, our transcriptomic data analysis revealed significant regulation of genes associated with ABA and JA signaling, plant cell wall composition, and ROS in FaWRKY29 or FaWRKY64 knockdown strawberry fruits in response to the pathogen. CONCLUSION: Our study uncovered the foundational role of WRKY transcription factor genes, FaWRKY29 and FaWRKY64, in conferring resistance against B. cinerea. The discovery of susceptibility genes involved in BFR presents significant potential for developing resistance breeding strategies in cultivated strawberries, potentially leveraging CRISPR-based gene editing techniques.

Pathogenicity and fungicide sensitivity of Phytophthora parvispora, a new pathogen causing gummosis and root rot disease on citrus trees.

In 2021, pomelo (Citrus grandi) trees grown in Tuyen Quang and Phu Tho in northern Vietnam suffered from leaf yellowing, gummosis on stems, brown rot on fruit, and black rot on roots. Based on morphological and sequence analysis of the ITS and cox1 gene regions, the pathogen causing gummosis and root rot of citrus trees was identified as Phytophthora parvispora. Pathogenicity assays using mycelial plugs and zoospore suspension showed that P. parvispora induces disease symptoms on both the upper and lower parts of various citrus trees, including pomelo, orange (C. sinensis), and lime (C. aurantiifolia). This is the first report of P. parvispora as the causative agent of gummosis and root rot on various citrus trees in South-East Asia as well as in Vietnam. Further, P. parvispora was sensitive to all tested fungicides, including mancozeb, chlorothalonil, fosetyl aluminium, potassium phosphonate, and dimethomorph. These findings will have important implications for the effective management of gummosis and root rot disease of citrus trees.

Occurrence of Postharvest Fruit Rot of Mango Caused by Fusarium pernambucanum in China.

Mango (Mangifera indica L.) is one of the most important tropical fruits in the world, thanks to its pleasant taste, aroma and high nutritional value (Ibarra et al. 2015). In June 2021, Surveys were conducted in three agricultural markets (113°36'E, 23°11'N) of the Yuancun district in Guangzhou, China. Postharvest fruit rot was observed on mango (about 25% of the fruits showed disease symptoms). Black rot symptomatic lesions were observed on the fruit surface and eventually penetrated the mesocarp of mango fruits. To isolate and identify the pathogen, fruits (n=35) were surface disinfected with 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water. Thirty small pieces (3-5 mm2) were excised from the lesion margins. The excised tissue pieces were cultured on potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips onto fresh PDA. Fungal isolates XTM-5 and XTM-8 were isolated from diseased fruits. All isolates grown on PDA had abundant, fluffy, whitish to yellowish aerial mycelia, and the colony reverse was pale brown. Macroconidia were falcate, slightly curved with 5-7 septa, 29.5-42.2 × 4.3-6.2 µm. Spindle-shaped mesoconidia were abundantly produced, straight to slightly curved with 3-4 septa, 20.3-24.5 × 4.6-4.8 µm. Microconidia were pyriform to obovate with 0-2 septa, 7.3-11.7 × 2.4-3.2 µm. Chlamydospores were globose or irregular, in chains and, hyaline to light brown. Based on the morphological characteristics, the fungus was tentatively identified as Fusarium pernambucanum (Santos et al. 2019). The molecular identity of the isolates was confirmed by sequencing the internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF1) and RNA polymerase subunit II gene region (RPB2) genes (White et al. 1990; O'Donnell et al. 2022). Sequences of isolate XTM-8 were deposited in GenBank (ITS: ON413679.1, TEF1:ON420221.2, RPB2: ON420222.2). A maximum-likelihood phylogenetic tree based on the concatenated sequences confirmed the isolates as F. pernambucanum (Xia et al. 2019). A pathogenicity test was conducted on mango. Six healthy fruits were inoculated with F. pernambucanum mycelial discs (5 mm in diameter) after being wounded with a needle or unwounded, six control fruits were inoculated with PDA agar. All inoculated fruits were incubated in the dark at 26°C and 95% relative humidity for 7 days post inoculation. Control fruits remained asymptomatic, whereas inoculated fruit developed symptoms on the fruit surface at the point of inoculation. The pathogenicity test was performed three times. The original isolates were confirmed morphologically and molecularly after they were reisolated from the symptomatic fruit, thus confirming Koch's postulates. F. pernambucanum is a widespread pathogen that causes diseases across a wide range of plant hosts in China, such as muskmelon fruit rot (Zhang et al. 2022); mango leaf spots (Guo et al. 2021) and plum leaf blight (Lu et al. 2022). To our knowledge, this is the first report of F. pernambucanum causing fruit rot of mango in China. As mango contamination with Fusarium mycotoxins poses a health risk for consumers, the occurrence of this disease needs to be carefully monitored to ensure effective disease management strategies are implemented in mango production.

First report of Calonectria ilicicola causing fruit rot on postharvest Prunus persica in Zhejiang Province, China.

Peach (Prunus persica) is an important economic tree fruit in China, with 15 million tons produced in 2020 (Xu et al. 2022). In September 2021, fruit rot on postharvest P. persica 'Yingqingtao' was observed in an orchard warehouse in Qixing district (120°41'E, 29°15'N), Zhejiang Province. Disease incidence was estimated at 25%, and yield loss was estimated at approximately 20% of the total yield. The naturally infected fruit had water-soaked, light brown lesions that fused, and produced a gray-white, dense mycelium (Fig. 1 A). The mycelia were transferred using a sterilized toothpick to potato dextrose agar (PDA) and cultured for 7 d. Macroconidia were used to produce five single-spore isolates, each from a different fruit. Six-day-old colonies grown on PDA at 26°C had light brown centers with gray-white edges; on the underside the centers were reddish brown and white towards the margin (Fig. 1 D). Isolate TGF2 was selected for further identification. Macroconidia were hyaline, straight, cylindrical, and one-to-three septae, 63.2 to 81.8 × 5.7 to 7.8 µm (mean = 73.9 ± 4.3 × 6.9 ± 0.5, n = 30) (Fig. 1 E). Chlamydospores were produced abundantly on PDA (Fig. 1 F), and measured 11.7 to 19.4 × 8.5 to 16.9 µm (n = 10). Perithecia were reddish orange, globose, and 329.9 to 417.1 µm in diameter on PDA (Fig. 1 G). Asci were hyaline and clavate, 61.2 to 91.8 × 14.4 to 20.7 µm (n = 10); ascospores were hyaline, slightly curved, 1- to 3-septate, mostly 1-septate, and 37.6 to 59.7 × 4.9 to 6.4 µm (mean = 49.9 ± 4.5 × 5.6 ± 0.4, n = 30) (Fig. 1 H-J). Morphological characteristics placed this organism within the Ca. kyotensis species complex (Liu et al. 2020). For molecular identification, the internal transcribed spacer (ITS: OP164807-OP164811), calmodulin (Cal: OP176049-OP176053), histone3 (His3: OP176054-OP176058), and translation elongation factor 1α (Tef1: OP176044-OP176048) genes were sequenced (Liu, et al., 2020). The twenty sequences were deposited in GenBank. A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype Ca. ilicicola CMW 30998 (Liu et al. 2020). Bayes phylogenesis suggested that these strains and Ca. ilicicola CMW 30998 were clustered in the same clade (Bayesian posterior probability = 1) (Fig. 2). Integrating morphology and molecular data, these strains were identified as Ca. ilicicola. For pathogenicity tests, P. persica fruits were surface sterilized in 75% ethanol for 30 s and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (30 mL of 1 × 106 conidia/mL) of TGF2 was sprayed onto ten fruits, and ten fruits sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 26°C and 60% relative humidity. After 5 days, inoculated fruits showed necrotic lesions and a dense, gray-white mycelium, however, the control fruits showed no symptoms (Fig. 1 B, C). Ca. ilicicola was reisolated from lesions of inoculated fruits. Ca. ilicicola has been reported from Vaccinium sp., Glycine max, Medicago sativa (Farr and Rossman 2022; Kleczewski et al. 2019; Zhang et al. 2020). To our knowledge, this is the first report of Ca. ilicicola causing fruit rot of P. persica in China. In other research on Ca. ilicicola, we found that continuous light could inhibit its growth, suggesting a method to protect postharvest peaches.

First Report of Muskmelon Fruit Rot Caused by Fusarium sulawesiense in China.

Muskmelon (Cucumis melo L.) is a widely cultivated and economically important fruit crop worldwide. In June 2022, fruit rot symptoms were observed on ripening muskmelons (cv. Boyang) in Shouguang City (36.81°N 118.90°E) of China. To determine the causal agent, we surveyed 200 muskmelon plants in about 1000 m2 of planting area and collected diseased muskmelons. Approximately 20% of muskmelon fruits had symptoms, and yield loss averaged 20%. Water-soaked lesions were observed on the surface and the fruit rotted from inside. Lesions were covered with white mycelium. Rotted fruit were surface-disinfested with 1% NaOCl for 1 min, 75% ethanol for 30 s, and washed three times with sterile water. Pieces (1 cm3) were cut from the disinfested fruit, placed on potato dextrose agar (PDA), and incubated at 25°C for 1 week. Ten isolates with similar morphology were obtained and isolates SG66 and SG68 were selected for further characterization. Colonies maintained on PDA in the dark had an average radial growth rate of 10-12 mm/d at 25°C. Surface was white, velvety to felty mycelium. Reverse was white to pale wheat. Diffusible pigments were absent. On carnation leaf agar, sporodochia appeared as slimy dots, macroconidia were 3- to 5-septate, 20-35 × 3-5 µm, falcate, with a pronounced dorsiventral curvature, with blunt to papillate apical cell, and barely to distinctly notched basal cell. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with descriptions of Fusarium sp. DNA was extracted from isolates SG66 and SG68 using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using generic primers, the products sequenced, and sequences deposited in GenBank (ITS: OP251362, OP251363; CAM: OP266024, OP266025; RPB2: OP266028, OP266029; TEF1: OP266026, OP266027). Isolates SG66 and SG68 clustered with Fusarium sulawesiense (85% bootstrap) (Maryani et al. 2019). The Fusarioid-ID database pairwise alignment of ITS (526 bp), CAM (534 bp), RPB2 (861 bp), and TEF1 (636 bp) sequences from isolate SG66 showed 99.6% (98.9% coverage), 100% (100% coverage), 100% (100% coverage) and 100% (98.4% coverage) similarity with the corresponding sequences (GQ505730, LS479422, LS479855 and GQ505641), respectively, of the reference strains of F. sulawesiense (InaCC F940 and NRRL 34059). To perform a pathogenicity test, 10 µl of conidial suspensions (1 × 106 conidia/ml) were injected into ten muskmelon fruit using a syringe, and ten control fruit were inoculated with 10 µl of sterile distilled water. The test was repeated three times. After 7 days at 25°C, the pulp of all inoculated muskmelons began to rot, and the lesion expanded from the inside to the fruit surface at the injection site and became covered with white mycelia. No symptoms developed on the control fruit. The fungus was successfully re-isolated from infected tissues and confirmed as F. sulawesiense by morphological and phylogenetic analyses. F. sulawesiense has previously been reported on yellow melon (Canary) in Brazil (Lima et al. 2021) and on a range of hosts, including Luffa aegyptiaca, in China (Wang et al. 2019). To our knowledge, this is the first report of muskmelon fruit rot caused by F. sulawesiense in China.

First Report of Fruit Rot Caused by Fusarium luffae in Cherry Tomato in China.

Tomato (Solanum lycopersicum L.) is a fruit of great economic value that is grown worldwide. In November 2022, fruit rot symptoms were observed in cherry tomatoes (cv. Qianxi) in Jinan City of Shandong Province, China. Six cherry tomato samples (four symptomatic and two asymptomatic) were collected from commercial fields (approximately 1.2 ha) where the incidence of the disease ranged from 5 to 10%. The core and surface of the infected fruit were colonized and covered with white mycelia. Tissue pieces (5 mm × 5 mm) from the junction of healthy and diseased samples were surface-disinfected with 75% ethanol for 3 min, followed by 10% sodium hypochlorite for 5 min, and washed three times with sterile water. Tissue pieces were cultured on potato dextrose agar (PDA containing 200 mg/L timentin) at 28°C for five days. Four fungal isolates with similar morphological characteristics were obtained from each sample. Two representative isolates were collected and purified using the single-spore method. After five days on PDA at 28°C, FL1 and FL2 colonies showed abundant white to cream colored aerial mycelia with an average growth rate of 5 mm/day. On carnation leaf agar, FL1 was characterized by falcate macroconidia with pronounced dorsiventral curvature containing three to eight tapered apical cells and foot-shaped basal cells ranging in size from 25 to 74 µm × 3.6 to 6.8 µm (n=50). Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of F. luffae (Wang et al. 2019). DNA was extracted using the CTAB method. The nucleotide sequences of the translation elongation factor 1-α gene (TEF1) and the second largest RNA polymerase II subunit (RPB2) were amplified using specific primers EF1/EF2 and RPB2F/R, respectively (O'Donnell et al. 1998, 2010). FL1 and FL2 sequences were deposited in GenBank (TEF1: OQ427345 and OQ427346, RPB2: OQ427347 and OQ427348). Polyphasic identification indicated 100% similarity of FL1 and FL2 to F. luffae. A combined dataset of TEF1 and RPB2 was aligned using MAFFT v.7, and phylogenetic analysis was performed in MEGA v.7.0 using the maximum likelihood method. The cherry tomato isolates (FL1 and FL2) clustered together with the F. luffae reference strain NRRL31167 (100% bootstrap) and were identified on a morphological and molecular basis as F. luffae belonging to the Fusarium incarnatum-equiseti species complex. F. luffae was the only pathogen recovered from the infected fruit. To test for pathogenicity, healthy cherry tomato fruit were inoculated in a greenhouse (28°C, 12/12 h light/dark cycle, 90% relative humidity), six by wounded inoculation and six by nonwounded inoculation) with 10 µL conidial suspensions of isolate FL1 at 1 × 106 conidia/mL. Six wounded-treated cherry tomato fruit were used for the control. All cherry tomatoes were kept in a growth chamber at 28℃ with 90% relative humidity. After seven days, the inside of the wound inoculated fruit began to rot, expanding toward the surface and producing white mycelia. Two diseased cherry tomatoes were randomly selected for tissue isolation and F. luffae was re-isolated showing the same morphology as the FL1 isolate, thus fulfilling Koch's postulates. The nonwounded inoculated fruits and control cherry tomatoes remained asymptomatic with no pathogens recovered. This indicates that the wound is an important way for F. luffae to invade tomato, and fruit rot is caused by F. luffae's infection of tomato. To the best of our knowledge, F. luffae has caused fruit rot in muskmelon (Zhang et al. 2022), but this is the first report of fruit rot disease in cherry tomatoes caused by F. luffae in China. Since cherry tomatoes are an important commercial crop in China, F. luffae infection has the potential to pose a threat to the industry.