L. P. Liu

A Novel MFS Transporter Gene ChMfs1 Is Important for Hyphal Morphology, Conidiation, and Pathogenicity in Colletotrichum higginsianum

Colletotrichum higginsianum is a widely distributed fungus attacking many cruciferous species. To investigate pathogenic mechanisms of the pathogen on the host Arabidopsis thaliana, we screened and obtained a virulence-deficient mutant Ch-1-T513 in a T-DNA insertion mutant library of C. higginsianum. The mutant Ch-1-T513 produced yellow colony centers with distorted multi-branching hyphal tips as well as producing few conidia. Heavily swollen hyphae in the mutant could be observed, and intra-hyphal hyphae were found to be formed in the balloon-shaped hyphae. The mutant failed to produce lesions on 12-day-old Arabidopsis seedlings, and invasive hyphae did not differentiate into large primary and thin secondary hyphae after appressorial formation on Arabidopsis leaves, but formed abundant bulbous hyphae in epidermal cells. Southern blot analysis showed Ch-1-T513 had double-site T-DNA integrations. The mutant had insertions upstream of genes for a major facilitator superfamily (MFS) transporter, ChMfs1 and an aldo/keto reductase, ChAkr. Complementation experiments by transforming genomic sequences from a wild-type strain into the insertion mutant demonstrated that ChMfs1 is involved in the Ch-1-T513 phenotype. The complementation strain C-ChMfs1-1 exhibited normal hyphal morphology, conidiation, and pathogenicity identical to the wild-type. The results demonstrate that ChMfs1 is involved in intra-hyphal hyphae production, conidiation, and pathogenicity in C. higginsianum. To our knowledge, this is the first report of a MFS transporter gene in a phytopathogenic fungus associated with intra-hyphal hyphae formation, playing a key role in infection of its plant host.

First Report of Botrytis pelargonii Causing Postharvest Gray Mold on Fresh Ginseng Roots in China

Chinese ginseng (Panax ginseng) is a perennial Chinese herb of the Araliaceae family. It is found in the three northeast provinces of China. As a valuable restorative tonic, it is widely used in traditional Chinese medicine (Radad et al. 2006). In November 2015 and 2016, gray mold was observed on 10 to 35% of fresh ginseng roots in two storage facilities under complete darkness at 4°C in Ji An and Fu Song City, Jilin Province. Initial symptoms included light brown, water-soaked lesions on ginseng roots. Within 30 days, the diseased areas enlarged, with dense, abundant gray mycelia forming on lesions. To isolate the pathogen, small pieces of diseased roots were surface disinfested in 3% NaClO for 2 min, rinsed twice in distilled water, plated on potato dextrose agar (PDA), and incubated at 22°C for 5 days. Sixteen isolates were recovered by single-spore isolation. Colony morphology was almost identical among all isolates. Representative isolate PG-S015 was grown on PDA under constant fluorescent light at 22°C. After 5 days, abundant mycelia with branched conidiophores were produced. Conidia were single, aseptate, pale brown, and globose to subglobose. Conidial size ranged from 8.2 to 17.3 μm in length and from 5.8 to 12.2 μm in width, averaging 13.6 ± 0.6 × 8.7 ± 0.3 μm (n = 50). The conidia were slightly longer than B. cinerea (Zhang et al. 2006). After 9 days, black, scattered, irregular sclerotia were produced. Sclerotial size measured 1.0 to 3.0 × 1.3 to 6.0 mm with the average of 2.1 × 3.8 mm (n = 50). DNA was extracted from all isolates. All 16 isolates were sequenced for the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), DNA-dependent RNA polymerase subunit II (RPB2), and heat-shock protein 60 (HSP60) genetic markers. Their ITS sequences (KY810515 to KY810530) were identical to those of B. pelargonii (AJ716290) and B. cinerea (KY319171). The G3PDH (KY827182 to KY827197) and HSP60 (KY827198 to KY827213) sequences were 137 and 42 bp shorter than those of B. cinerea (GenBank KY275256 and KX229753) and identical to those of B. pelargonii (AJ704990 and AJ716046). The RPB2 sequences (KY827214 to KY827229) were also 99% identical to that of B. pelargonii (AJ745662). The fungus was identified as B. pelargonii based on its morphological and molecular characteristics (Staats et al. 2005; Zhang et al. 2006). Pathogenicity tests were performed by pipetting a 100 μl of conidial suspension (1 × 10⁵ conidia/ml) of each selected isolate PG-S015 and PG-S-FS-017 onto a 5-year-old fresh ginseng root, three replicates per isolate. Sterilized water was used as the noninoculated control treatment. The experiment was carried out three times. All inoculated roots were maintained at 4°C and 90% relative humidity, similar to the environmental conditions in which the disease was observed. Within 30 days, all inoculated ginseng roots developed similar symptoms to those of the naturally occurring disease described above, whereas all noninoculated roots remained healthy. Isolates with identical morphological and molecular characteristics were recovered from the diseased roots. Prior to this report, B. pelargonii was exclusively recovered from Pelargonium spp. (Strider 1985). This is the first report of B. pelargonii causing postharvest gray mold on stored fresh ginseng roots. Proper management should be taken to reduce the losses from this disease to the quality and marketability of ginseng as a highly valuable traditional Chinese medicine.