Microdochium chrysopogonis W. Zhang & X. Lu, 2023

Lu, Xiang, Mai, Mengxian, Tan, Wenhui, Zhang, Muyan, Xie, Jie, Lu, Yi, Niu, Xue Li & Zhang, Wu, 2023, Identification and fungicide sensitivity of Microdochium chrysopogonis (Ascomycota, Amphisphaeriaceae), a new species causing tar spot of Chrysopogon zizanioides in southern China, MycoKeys 100, pp. 205-232 : 205

Microdochium chrysopogonis W. Zhang & X. Lu sp. nov.

Figs 1 , 3

Etymology.

Name refers to Chrysopogon , the host genus from which this fungus was collected.

Description.

Sexual morph on infected leaf tissue of the host plant (CAF 800054). Ascomata perithecial, 300-350 μm diam., solitary or in groups, immersed, pale brown to black, subglobose to oval, uniloculate, non-ostiolate. Paraphyses filiform, hyaline, straight or curved, apically free. Asci 50-60 × 10-18, x ¯ = 55 × 13 μm (n = 50), hyaline, fasciculate, unitunicate, oblong to narrowly clavate, fusiform, 8 biseriate spores with a short stipe. Ascospores clavate, hyaline, guttulate, 20-22 × 8-11.5, x ¯ = 21 × 9 μm (n = 50), aseptate, smooth. Sporodochia salmon-pink, slimy. Conidiophores reduced to conidiogenous cells. Conidiogenous cells with percurrent proliferation, hyaline, smooth, aseptate, ampulliform or obpyriform, 10-23 × 8-11.5, x ¯ = 17 × 9.5 μm (n = 50). Conidia fusiform, lunate, curved, solitary, guttulate, variable in length, 0-1-septate, 18-72 × 2-3.5, x ¯ = 38.5 × 3 μm (n = 50), apex rounded, base usually flattened. Chlamydospores not observed. Vegetative hyphae on PDA (GDMCC 3.683) superficial and immersed, septate, branched, hyaline, smooth, 1-5.5 μm wide.

Culture characteristics.

Colonies on PDA reaching 4.0-4.5 cm within seven days in the dark at 30 °C, flat, white cottony aerial mycelium, dense, saffron rounded sporodochia produced after 3 weeks; reverse saffron. On MEA, sparse white cottony aerial mycelium, orange rounded sporodochia produced; reverse salmon-pink. On OA, periphery with white scarce cottony aerial mycelium, concentric rings of orange rounded sporodochia produced; reverse orange.

Type.

China, Guangdong Province, Zhanjiang City, field of the Grass Research Station of Lingnan Normal University (LNU), from a leaf of vetiver grass ( Chrysopogon zizanioides ) with leaf tar spot disease, September 2019, W. Zhang & X. Lu, holotype CAF 800054, ex-type living strain GDMCC 3.683 .

Additional materials examined.

China, Guangdong Province, Zhanjiang City, field of the Grass Research Station of Lingnan Normal University (LNU), from a leaf of vetiver grass ( C. zizanioides ) with leaf tar spot disease, September 2019, W. Zhang & X. Lu, strain LNU-196 ; China, Guangdong Province, Zhanjiang City, field of the Grass Research Station of Lingnan Normal University (LNU), from a leaf of vetiver grass ( C. zizanioides ) with leaf tar spot disease, September 2019, W. Zhang & X. Lu, strain LNU-197 .

Notes.

A multilocus phylogenetic analysis of the ITS, LSU, tub2 and rpb2 loci placed three strains of M. chrysopogonis in a distinct and monophyletic clade (1/100) sister to M. dawsoniorum and M. ratticaudae . Notably, M. chrysopogonis has longer conidia (18-72 × 2-3.5 μm) than M. ratticaudae (7-11 × 1.5-2.5 μm) and wider conidia than M. dawsoniorum (25-75 × 1-2 μm). Furthermore, the conidia of M. chrysopogonis are guttulate and 0-1-septate, while those of M. dawsoniorum are 0-3-septate and those of M. ratticaudae are aseptate. The conidiogenous cells of M. chrysopogonis appear as percurrent, ampulliform or obpyriform, whereas those of M. ratticaudae are indistinct from the hyphae and those of M. dawsoniorum are cylindrical to irregular and flexuous. Additionally, the conidiogenous cells of M. chrysopogonis (10-23 × 8-11.5 μm) are wider than those of M. ratticaudae (20-30 × 1-2 μm) (Table 3 ). Differences are also evident in the sexual morph of these three species. In particular, the sexual morph is not observed in M. dawsoniorum . Ascomata size varies, with that of M. ratticaudae (100-160 μm) being smaller than that of M. chrysopogonis (300-350 μm). Ascospores of M. ratticaudae (14-24 × 4-7 μm) are fusoid to navicular, while those of M. chrysopogonis are clavate, guttulate and wider (20-22 × 8-11.5 μm). In addition, M. ratticaudae features abundant, pale to olivaceous brown, subglobose or cylindrical chlamydospores, while these are not observed in M. chrysopogonis ( Crous et al. 2020, 2021a; Table 3 ). Consequently, based on both morphological characteristics and phylogenetic analyses, all three isolates of M. chrysopogonis were proposed as a new species.

Pathogenicity test

The symptoms observed on leaves of C. zizanioides after inoculation with the representative isolate GDMCC 3.683 were similar to those observed in the field. No symptoms were observed on the leaves of the negative controls (Fig. 4 ). The average disease incidence of detached leaves that were wounded and sprayed with the isolates GDMCC 3.683, LNU-196 and LNU-197 was 93.3%, 80.0% and 93.3%, respectively. The average disease incidence of whole plants after spraying with the same isolates was 76.7%, 73.3% and 73.3%, respectively (Fig. 5 ). Koch’s postulates were fulfilled by successful re-isolation of the fungal strains from all leaf spot tissues inoculated with the three isolates. The morphology and DNA sequences of the isolates re-isolated from the inoculated tissues were consistent with those of the strains used for inoculations.

Effect of temperature on mycelial growth

The mycelial growth of M. chrysopogonis was significantly affected by temperature (P <0.01). All three isolates of M. chrysopogonis grew between 10 and 40 °C, with maximum growth observed at 30 °C (Fig. 6 ). No isolates grew at 5 or 45 °C after 3 days. The highest average mycelial growth rate was observed at 30 °C (26.5 ± 2.0 mm/day), followed by 25 °C (20.1 ± 4.7 mm/day).

Fungicide sensitivity

The EC50 values of various fungicides were analysed for their effectiveness against M. chrysopogonis isolates. A total of 17 isolates of M. chrysopogonis were collected from diseased leaves spanning the period from 2019 to 2022.

The frequency distribution showed that difenoconazole, fludioxonil, flusilazole, carbendazim and iprodione exhibited distributions resembling normal curves, while pyrimethanil, propiconazole, metalaxyl and tebuconazole displayed unimodal curves (Fig. 7 ). EC50 values for the inhibition of 17 M. chrysopogonis isolates, based on mycelial radial growth, varied across fungicide treatments (P <0.05) (Table 4 ). Amongst the tested fungicides, flusilazole had the lowest EC50 values, with a notably concentrated response range of 0.001 to 0.007 µg /ml and an average of 0.004 µg /ml. Tebuconazole closely followed with a slightly narrower range, exhibiting values ranging from 0.002 to 0.009 µg /ml and an average of 0.007 µg /ml. Furthermore, there was no significant difference between flusilazole and tebuconazole. Propiconazole displayed EC50 values spanning from 0.006 to 0.016 µg /ml, with an average of 0.011 µg /ml, while those of carbendazim ranged from 0.008 to 0.031 µg /ml, with an average of 0.024 µg /ml. In contrast, those of difenoconazole exhibited a broader range, varying from 0.013 to 0.127 µg /ml, with a mean value of 0.077 µg /ml, while those of pyrimethanil ranged from 0.054 to 0.605 µg /ml, with an average of 0.411 µg /ml. Those of iprodione, on the other hand, spanned from 15.018 to 260.335 µg /ml, with an average of 193.031 µg /ml. Metalaxyl exhibited the highest EC50 value, displaying the widest range amongst all fungicides, extending from 302.785 to 1056.896 µg /ml with an average of 892.677 µg /ml. Overall, these findings indicate varying degrees of sensitivity to different fungicides amongst M. chrysopogonis isolates. These variations in sensitivity could be essential considerations for designing effective fungicide application strategies against vetiver leaf tar spot disease.

The inhibition of mycelial growth revealed that all nine fungicides exhibited a reduction in fungal growth in vitro when compared to plates without amendments. The effectiveness of these fungicides in diminishing the mycelial growth of the isolates was contingent upon both the specific chemical compound and its concentration. Four DMI fungicides, namely, difenoconazole, propiconazole, flusilazole and tebuconazole and one MBC fungicide, carbendazim, displayed strong activity against M. chrysopogonis growth at concentrations below 1 µg /ml, specifically at concentrations of 1, 0.2, 0.1, 0.2 and 0.2 µg /ml, respectively (Fig. 8 ). However, M. chrysopogonis showed a tendency to exhibit better growth in the presence of pyrimethanil, fludioxonil, iprodione and metalaxyl, with mycelial growth being completely inhibited at concentrations exceeding 100 µg /ml (Fig. 8 ).

Figures

Figure 1. Disease symptoms and morphological characters of Microdochium chrysopogonis on infected leaf tissue (CAF 800053) A-C tar spot symptoms of Chrysopogon zizanioides from natural infection in the field D appearance of immersed ascomata on infected leaves E ascomata in longitudinal section F-H asci I-N ascospores. Scale bars: 100 μm (D, E); 10 μm (F-H).

Figure 3. Microdochium chrysopogonis (from ex-type: GDMCC 3.683) A colonies after 7 days on PDA B colonies after 7 days on MEA C colonies after 7 days on OA D colony overview of the sporodochia on PDA in culture after incubation for three weeks E aggregated conidiophores F conidiophores with conidiogenous cells G, H conidia. Scale bars: 20 μm (B).

Figure 4. Tar spot symptoms of Chrysopogon zizanioides 7 days after spraying on detached leaves (A, B) and 10 days after spraying on leaves attached to whole plants (C, D) with Microdochium chrysopogonis isolate GDMCC 3.683 (B, D) and sterilised water (A, C).

Figure 5. Disease incidence of tar spot symptoms on Chrysopogon zizanioides for leaves 7 days after spraying detached leaves (A) and for whole plants 10 days after spraying leaves attached to potted plants (B), respectively, with Microdochium chrysopogonis isolates GDMCC 3.683, LNU- 196 and LNU- 197. Values are shown as the means, with the error bars representing the standard error. For each pathogen, columns with the same letter indicate means that are not significantly different according to a least significant difference (LSD) test (P <0.05).

Figure 6. Colony growth rate of three isolates, GDMCC 3.683, LNU- 196 and LNU- 197, of Microdochium chrysopogonis from Chrysopogon zizanioides under different temperatures. Error bars represent the standard error.

Figure 7. Frequency distribution of the 50 % effective concentration (EC 50) values of six groups including nine fungicides for Microdochium chrysopogonis isolates, based on mycelial growth from 2019 to 2022.

Figure 8. Effect of fungicides on the mycelial growth of Microdochium chrysopogonis. Values are shown as the means, with the error bars representing the standard error.