Field symptom survey and fungal isolation
In 2017, the novel occurrence of zedoary turmeric (Curcuma phaeocaulis) leaf blight emerged within the planting area of Nanxiong village, situated in Guilin city, Guangxi Province. The pathogenic impact was confined exclusively to the foliage, evoking discernible symptoms exclusively at the tips and margins of the upper and central leaves. During the initial stages of the disease progression, minute yellow‒green lesions manifested on the leaf surfaces (Fig. 1a). Subsequently, over a span of nearly 30 days, these lesion expanded toward leaf's tip or periphery, assuming a central brown hue with a yellowish border (Fig. 1b). A period of two months saw the gradual spread of these lesions to encompass the entirety of the leaf. Color transitions were noted from deep yellow at the periphery, evolving sequentially through pale brown to dark brown at the center. Concurrently, minuscule black speckles emerged on the lesion's surface. Clear demarcation materialized between afflicted and unaffected leaf regions. The ailment's exacerbation ultimately led to the demise of the entire plant (Fig. 1c).
Pathogenicity test
The isolate CP1 caused wilt on the turmeric leaves, similar to natural infection. At 24 h after the wound inoculation of the strain CP1, the lesions were waterlogged in the early stages, and the boundary between disease and health was obvious (Fig. 2a, b). Under the same experimental conditions, the leaves also showed light brown lesions 48 h after non-wound inoculation (Fig. 2c, d). All the inoculated leaves showed the same symptoms as those observed in the original disease. The pathogen was then re-isolated from the legions, which was consistent with the inoculated pathogen. There were no symptoms in the negative controls.
Phylogenetic analysis
The alignment comprised 108 strains (including the outgroup taxon Pestalotiopsis and Pseudopestalotiopsis) and the manually adjusted dataset comprised 1568 characters (including 490 bases for ITS, 654 bases for TUB, 424 bases for TEF1-α). Suitable models were selected using models of nucleotide substitution for each gene. The HKY+I+G model with gamma-distributed rate model for ITS and the K80+G model with gamma-distributed rate model for TUB and the K80+I+G model with gamma-distributed rate model were selected for TEF and included for each gene partition. Although the strain closely related to N. brasiliensis (MFLUCC 17-555) and N. surinamensis (CBS 450.74), support among them was low, and there were differences in pairwise nucleotide. Therefore, we speculate that CP1 is a novel Neopestalotiopsis strain (Fig. 3).
Morphological analysis
The isolate were procured from ten infected samples, subsequently undergoing morphologically evaluation concerning colony color, sporulation, and conidial morphology on artificial growth media. Under the conditions of incubation at 25℃ for 7 days, the cultural features observed on PDA plates comprised of an opulent growth of aerial mycelia, forming petal-like colonies (Fig. 4a). Notably, the upper surface of these colonies exhibited a pale-yellow hue, while the reverse displayed prominent radial grains, rendering a white appearance (Fig. 4b). The conidiomata, with a diameter ranging from 50-100 μm, emerged as acervulus structures, originating subepidermally and featuring basal stromata. The conidiogenous cells were distinct, simplistic, diminutive, and filiform in nature (Fig. 4c). Subsequently, the conidia manifested as spindle-shaped structures, tending to be upright or gently curved, characterized by 4 septations and 5 cellular units (Fig. 4d). Among these, two terminal cells were transparent, while the three intermediary cells exhibited a light brown hue, albeit with subtle color variations. The conidial dimensions spanned from 18.6 to 33.8 μm in length and 2.6 to 7.6 μm in width (`х = 24.95 × 5.24). The median cell trio measurement was 12.4–19.8 (`х = 17.05) μm (Fig. 4e-f). The terminal cells, both apical and basal, tapered towards their ends and were transparent. Specifically, the apical cells ranged from 2.4 to 7.1 μm in length (`х = 4.71 μm), while the basal cells spanned from 3.5 to 6.8 μm (`х = 4.89 μm). Each conidium exhibited the formation of 2 to 4 (predominantly 3) apical appendages and a solitary basal appendage. The apical appendages measured between 11.9 and 28.8 μm (`х = 20.6 μm), while the basal appendage ranged from 2.5 to 13.6 μm (`х = 5.9μm) in length (Fig. 4h). Therefore, both colony and conidial morphology differences among the strain CP1 with the species N. brasiliensis and N. surinamensis were compared. (Table 2). Based on molecular and morphological evidences, isolates obtained in this study were identified as Neopestalotiopsis strain CP1 a novel species.
Effect of temperature on the Neopestalotiopsis strain CP1
Temperature had a significant effect on the radial mycelial growth and spore formation of the isolate CP1 (P < 0.05). The isolates were able to grow from 5 to 35°C and produce conidia from 10 to 35°C on PDA (Fig. 5a, b). Twenty-five centigrade was the optimum growth temperature for the strain CP1, with hyphal growth rate of 11.5 mm/d and spore amount of 17.2×106 conidia.
Effect of light on the Neopestalotiopsis strain CP1
Radial mycelial growth and conidia formation of the strain CP1 on PDA were significantly affected by illumination (P < 0.05). Eighteen hours of light plus 6 h of darkness significantly promoted hyphal growth, with a growth rate of 11.9 mm/d (Fig. 6a). Conidial production under 24 h illumination was significantly higher than that under the other treatments, reaching 8.54×106 conidia/plate, and the lowest production under 24 h darkness was 0.35 × 106 conidia/plate (Fig. 6b).
Metabolism analysis
The metabolic capacity of Neopestalotiopsis strain CP1 was analyzed using phenotype microarray (PM) plates 1–5, 9 and 10 (Supplementary Table S2). Using data from PM1 and PM2 (carbon sources), the total carbon source utilization rate of the strain CP1 is 31.57%, and 60 kinds of substances can be effectively utilized. In addition, 14 carbon sources can promote the growth and metabolism of the strain, including D-galactose, dulcitol, D-mannitol, α-D-glucose, sucrose, gentiobiose, D-melezitose, maltotriose, M-inositol, tyramine, laminarin, pectin, butein, and D-raffinose. Using the PM3 plate, the strain CP1 was tested for its ability to grow on 95 different nitrogen sources (amino acids). Approximately 35 compounds were effectively utilized by the pathogen, including guanine, L-tryptophan and agmatine. Using the PM4 plate, the strain CP1 was tested for its ability to grow on 59 different phosphorus compounds and on 35 different sulfur substrates. Only four phosphorus source substances can be effectively utilized by this fungus, including D-mannose-6-phosphate, O-phospho-D-tyrosine, O-phospho-L-tyrosine and D-mannose-1-phosphate. There are eight kinds of sulfur sources that can be utilized by the strain, among which tetramethylene sulfone is the substance with the highest utilization rate. Ninety-five different biosynthetic pathways in the strain CP1 were investigated using PM5 panels. It was found that only 2'-deoxyadenosine could be effectively metabolized and utilized by the strain, and other substances such as L-arginine, inosine, and D-alanine could not be metabolized and utilized by the strain.
According to the data of the PM9 plate, the metabolism of the strain CP1 is different under different ionic strengths and osmotic pressures. Under the conditions of 100 mM ammonium sulfate (pH 8), 4% sodium lactate, 5.5% NaCl, 200 mM sodium phosphate (pH 7), 100 mM sodium nitrate, and 80 mM sodium nitrate, the strain maintained high metabolic ability. The results of the PM10 plate data showed that the optimum pH of conidia germination of the pathogen was between 5.0 and 5.5. At pH 4.5, 36 compounds, such as L-glutamic acid, L-leucine and L-phenylalanine, were available to the strain, while at pH 9.5, only 14 compounds were available and underutilized.