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

Abstract

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.