First Report of Strawberry Root Rot Caused by Fusarium falciforme in China.

Root rot caused by Fusarium spp. is a destructive disease affecting agricultural regions worldwide. Strawberries (Fragaria × ananassa Duch.) are an economically important crop in China. In March 2023, root rot was observed in strawberries grown in Jinan, Shandong Province, China. Symptoms included leaf wilt, necrotic roots, and plant death (Figure 1). Four strawberry samples (two symptomatic and two asymptomatic) were collected from ~2-acre fields where the disease incidence rate ranged from 2 to 3%. Tissue pieces (5 mm × 5 mm × 5 mm) from two healthy and two diseased strawberry root tissues were surface-disinfected with 75% ethanol for 3 min, treated with 10% sodium hypochlorite for 5 min, and washed three times with sterile water. These pieces were cultured for 5 days at 28°C on potato dextrose agar (PDA) containing 200 mg/L timentin. Typical Fusarium spp. like growth was observed on plates with the two symptomatic samples. Two representative fungal isolates (CM1 and CM2) with similar morphological characteristics were purified using the single-spore method (Figure 1). CM1 showed an average growth rate of 5 mm/d in PDA and comprised of several white-to-cream aerial mycelia after 5 d. After cultivation in carnation leaf agar medium for 7 d, falciform macroconidia, with blunt apical cells and slightly hooked basal cells comprising 3 to 4 septa of varying sizes (20 to 39)×(3.6 to 6.7 µm) were observed (n=50) (Figure 1). The chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, and 3.1-10.5 µm in diameter (Figure 1). The microconidia on PDA were (5.8 to 13.6)× (2.5 to 3.3) µm in size (n=50). These morphological characteristics are consistent with previous descriptions of the Fusarium solani species complex (FSSC). DNA was extracted using the CTAB method (Stenglein and Balatti 2006). The internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified and sequenced using specific primers (O'Donnell et al. 2010). The ITS (OR526528, OR526529), tef1 (OR536947, OR536948), rpb1 (OR536949, OR536950), and rpb2 (OR536951, OR536952) sequences of the CM1 and CM2 isolates were uploaded to the NCBI database. BLASTn analysis revealed that the ITS, tef1, rpb1, and rpb2 sequences were 99.1-100% identical to those of the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978. A phylogenetic tree based on the ITS, tef1, rpb1, and rpb2 sequences was generated using MEGA v.11 via the maximum-likelihood method (Tamura et al. 2021). CM1 clustered with the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978 and belonged to the FSSC based on its morphological and molecular characteristics (Figure 2). To test for pathogenicity, the roots of nine 3-month-old healthy strawberry (cv. Akihime) plants were exposed to conidial suspensions (1×108 spores/mL) of the CM1 isolate. Another nine root samples were treated with sterile water and used as controls. All strawberry plants were maintained in a growth chamber under a 12/12 h light/dark cycle at 28°C and 90% relative humidity and the experiment was repeated three times. After one month, the inoculated plants had withered and died, and the pith became dark red (similar to field plants) (Figure 1). The fungi isolated from the experimental plants were confirmed as F. falciforme using morphological and sequence analyses. F. falciforme causes root rot in several species including Nicotiana tabacum (Qiu et al. 2023) and Weigela florida (Shen et al. 2020); however, this study is the first to report root rot caused by F. falciforme in strawberries in China. Overall, F. falciforme infection poses a threat to strawberry production and breeding.

Selection and Validation of Reference Genes in Virus-Infected Sweet Potato Plants.

Quantitative real-time PCR (qRT-PCR) in sweet potatoes requires accurate data normalization; however, there are insufficient studies on appropriate reference genes for gene expression analysis. We examined variations in the expression of eight candidate reference genes in the leaf and root tissues of sweet potatoes (eight nonvirus-infected or eight virus-infected samples). Parallel analyses with geNorm, NormFinder, and Best-Keeper show that different viral infections and origin tissues affect the expression levels of these genes. Based on the results of the evaluation of the three software, the adenosine diphosphate-ribosylation factor is suitable for nonvirus or virus-infected sweet potato leaves. Cyclophilin and ubiquitin extension proteins are suitable for nonvirus-infected sweet potato leaves. Phospholipase D1 alpha is suitable for virus-infected sweet potato leaves. Actin is suitable for roots of nonvirus-infected sweet potatoes. Glyceraldehyde-3-phosphate dehydrogenase is suitable for virus-infected sweet potato roots. The research provides appropriate reference genes for further analysis in leaf and root samples of viruses in sweet potatoes.

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.

First Report of Cucumber Green Mottle Mosaic Virus Infecting Zucchini (Cucurbita pepo) and Wax Gourd (Benincasa hispida) in China.

Cucumber green mottle mosaic virus (CGMMV) was first discovered in China in 2003 and caused an epidemic in 2005. In China, the virus has been reported in gourd crops including watermelons, cucumbers, melons, etc (Sui et al. 2019). In Shandong Province, China from September 2014 to 2017, approximately 30% of zucchini (Cucurbita pepo) and wax gourd (Benincasa hispida) plants in commercial cucurbit fields, the two most important cash crops, exhibited chlorosis, mosaic, and mottling symptoms suspected to be caused by a tobamovirus. To identify the causative pathogens, ten zucchini and 15 wax gourd samples were collected from the commercial cucurbit fields. Total RNA was extracted and all samples were tested using reverse transcription PCR (RT-PCR) with TobamodF/TobamodR and TobamodF2/TobamodR2 (Li et al. 2018a). Five common Cucurbitaceae viruses were also tested: cucumber mosaic virus, papaya ringspot virus, squash mosaic virus, watermelon mosaic virus, and zucchini yellow mosaic virus (Ali et al. 2012). All samples generated positive results using tobamovirus generic primers but were negative for the five common Cucurbitaceae viruses. Amplification products (880 bp) from all samples were inserted into pMD19-T and recombinant clones were selected for Sanger sequencing. The results showed that zucchini green mottle mosaic virus, CGMMV, and tobacco mosaic virus (TMV) were detected in zucchini samples. CGMMV and TMV were detected in the wax gourd samples. To confirm the presence of these viruses, RT-PCR was performed using specific primer pairs, including CGMMV-cpf/CGMMV-cpr (Chen et al. 2006), ZG-F/ZG-R (Li et al. 2018b), and TMV-CP-F/TMV-CP-R (Srivastava et al. 2015). CGMMV was detected in all samples, with four zucchini and nine wax gourds only containing CGMMV. Zucchini (n=4; CGZ1-CGZ4) and wax gourd (n=4; CGWX1-CGWX4) isolates were cloned into pMD19-T and sequenced bidirectionally. The BLASTn results confirmed the presence of CGMMV, and the sequencing results were processed using DNAMAN Version (Lynnon Biosoft, San Ramon, CA, USA) and submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/). A phylogenetic tree based on the CGMMV coat protein (CP) was constructed using CGZ1-CGZ4 (OP779762-OP779765), CGWX1-CGWX4 (OP779766-OP779769), and representative CGMMV sequences from GenBank. Sequence analysis of the CP demonstrated that CGMMV-zucchini and -wax gourd isolates belonged to an independent branch of the Chinese muskmelon AH-FT197 isolate (KU175639) and had 100% identity with the AH-FT197 isolate. To confirm their infectivity, leaf sap extract of CGZ4 and CGWX4 in phosphate buffer (0.1 M, pH 7.0) was mechanically inoculated on leaves of virus-free zucchini seedlings (Cucurbita pepo cv. Zaoqingyidai, 4-leaf-stage, n = 10) or virus-free wax gourd seedlings (Benincasa hispida cv. Tiezhu 2, n = 10). Ten days after inoculation, all plants exhibited symptoms (systemic chlorosis, mosaic, and mottling) similar to those of diseased plants in the field. Control seedlings inoculated with phosphate buffer remained symptomless. RT-PCR analysis using the CGMMV-cpf/CGMMV-cpr primer confirmed that all ten zucchini or wax gourd seedlings were infected with CGMMV, and all the control plants were free from CGMMV. To the best of our knowledge, this is the first report on zucchini and wax gourd as natural hosts for CGMMV in China. CGMMV is a highly contagious seed-borne virus and further attention should be paid to its spread in cucurbit crops.