First Report of Lotus Seedpod Withering Caused by Fusarium proliferatum on Nelumbo nucifera Plants in China.

Abstract

Nelumbo nucifera (Nymphaeaceae family) is a well-known plant in China and with the increasing value of this crop, the planting area of lotus is expanding. In May 2019, an unknown withering lotus seedpod was obtained in Guangchang County of Jiangxi Province (26.79°N, 116.31°E). The disease arose between May and July of each year, resulted in the withering and consequent death of ~10% of lotus seedpods, with the disease being most serious during the rainy season. The initial symptoms of this disease include the shrinking of young lotus seedpods with concomitant yellowing of the epidermal tissue layer. These pods failed to grow normally and could to wither and die within one week, with the withering symptoms gradually spreading to associated stem tissues. To characterize the pathogens responsible for this disease, ten diseases seedpods were collected and cut into pieces of ~5×5 mm, then sterilized with 75% ethanol for 30 s, and treated with 0.1% mercuric chloride for 5 min. After being washed four times under sterilized water, samples were then transferred onto potato dextrose agar (PDA) and incubated for 7 d at 28℃ in the dark. Eight purified isolates yielded large numbers of aerial mycelium that were initially white in color, but then changed to a purple-red color over the course of this incubation period. The average mycelial growth rate was 6.3 mm per day (n=5). On PDA, macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The microconidia were hyaline, ovoid or ellipse and 4.6-13.5×2.2-4.3 µm in size (average: 8.7×3.1 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). To confirm the identity of this putative pathogen at the molecular level, the universal ITS4/ITS5 primers (White et al., 1990), the Fusarium specific pair PRO1/PRO2 (Mulè et al., 2004), EF1T/EF2T (O'Donnell et a., 1998) and RPB2F/R (O'Donnell et al., 2010) primers were utilized to amplify the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-internal transcribed spacer 2 (ITS2), calmodulin, alpha elongation factor genes, and RNA-dependent DNA polymerase II subunit from these isolates. Following alignment of the resultant sequences with GenBank via a BLAST analysis, the sequences (GenBank accession numbers: MW862499, MW762531, MW767988, MW831311, respectively.) showed 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank accession numbers: MW817705, LS423443, MH153750, and MW091308, respectively.). Based upon these morphological and molecular findings, this pathogen was identified as F. proliferatum. Pathogenicity testing was then performed using five plump healthy lotus seedpods. Sterile needles were used to generate wounds (2 mm deep, 1 mm in diameter) a 10 µL suspension of prepared spores (1.0×106 spores/mL) derived from a 7-day-old culture grown on PDA was injected into the wound sites of the lotus seedpod. As a control, give seedpods were additionally wounded and injected as the same as treated with 10 µL of sterile water. The experiments were repeated three times with five biological replicates. All seedpods were then incubated at 28℃ in a growth chamber (12 h light/dark) with 80% relative humidity. After a 3-day incubation period, wounded sites injected with spore suspensions exhibited browning. Following a 5-day incubation period, a mean lesion diameter of 9.8 mm was observed, with white mycelia growing on the wound surface and with evident withering of the internal and external tissues near the wounded site. In contrast, blank control wound sites remained healthy. We were again able to isolate F. proliferatum from the infected lotus seedpods. Finally, eight isolates were obtained were identified as the pathogen based on these morphological and molecular analyses, thus fulfilling Koch's postulates. This is the first report to our knowledge to have described a case of F. proliferatum causing lotus seedpod withering in China, providing a foundation for future research efforts aimed at presenting diseases caused by this pathogen.