Fusarium hunanense Lin Huang, Jiao He & D.W. Li, 2024

He, Jiao, Li, De-Wei, Cui, Wen-Li, Zhu, Li-Hua & Huang, Lin, 2024, Morphological and phylogenetic analyses reveal three new species of Fusarium (Hypocreales, Nectriaceae) associated with leaf blight on Cunninghamia lanceolata in China, MycoKeys 101, pp. 45-80 : 45

Fusarium hunanense Lin Huang, Jiao He & D.W. Li sp. nov.

Fig. 8

Etymology.

Epithet is named after Hunan Province where the type specimen was collected.

Holotype.

China, Hunan Province, Yiyang City, Heshan District, Henglongqiao Town, 28°27′24″N, 112°29′7″E, isolated from leaf spots of Cunninghamia lanceolata , May 2017, Wen-Li Cui, (holotype: CFCC 57574). Holotype specimen is a living specimen maintained via lyophilization at the China Forestry Culture Collection Center (CFCC). Ex-type (HN33-8-2) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Host/distribution.

From C. lanceolata in Henglongqiao Town, Heshan District, Yiyang City, Hunan Province, China.

Description.

Sexual state not observed. Asexual state: sporulation abundant from erect conidiophores formed on the agar surface or aggregated in sporodochia. Conidiophores in the aerial mycelium, mostly unbranched, rarely basally dichotomously branched, forming monophialides on the apices; phialides slender, subulate to subcylindrical, monophialidic, smooth, thin-walled, (29.6-)31.6-54.6(-74.1) × (2.0-)2.2-2.8(-3.0) μm, (mean ± SD = 43.1 ± 11.5 × 2.5 ± 0.3 μm, n = 17), with slight periclinal thickening at the tip and a short flared apical collarette. Sporodochia cream colored, produced on the surface of carnation leaves and PDA medium. Conidiophores in sporodochia (26.0-)29.3-39.1(-46.8) μm, (mean ± SD = 34.1 ± 5.1 μm, n = 39), irregularly branched, short stipitate, occasionally in whorls bearing terminal 2-4 monophialides; sporodochial phialides subulate to subcylindrical, smooth, thin-walled, (11.4-)15.5-22.1(-28.6) × (3.3-)4.0-5.2(-6.0) μm, (mean ± SD = 18.8 ± 3.3 × 4.6 ± 0.6 μm, n = 51), with periclinal thickening and a small, flared collarette. Sporodochial macroconidia cylindrical to falcate, gently curved, typically with a blunt and almost rounded apical cell and a barely notched foot cell, 3-6-septate, hyaline, smooth, thin-walled. Three-septate conidia: (22.1-)22.6-39.4(-54.7) × (5.0-)5.5-6.7(-7.4) μm, (mean ± SD = 31.0 ± 8.4 × 6.1 ± 0.6 μm, n = 11); four-septate conidia: (50.3-)54.4-68.2(-69.6) × (6.9-)6.9-7.7(-8.0) μm, (mean ± SD = 61.3 ± 6.9 × 7.3 ± 0.4 μm, n = 10); five-septate conidia: (51.8-)60.6-73.0(-78.2) × (6.4-)6.1-7.1(-8.5) μm, (mean ± SD = 66.8 ± 6.2 × 6.6 ± 0.5 μm, n = 31); six-septate conidia: (69.8-)70.7-77.7(-79.6) × (7.1-)7.5-8.3(-8.3) μm, (mean ± SD = 74.2 ± 3.5 μm × 7.9 ± 0.4 μm, n = 10). Chlamydospores developed in large numbers in hyphae and also in mature macroconidia. The chlamydospores were 0-1-septate, globose to ellipsoidal, constricted at the septum, intercalary or terminal in chains or solitary with mostly a pale color and smooth, (11.7-)11.7-12.9(-13.5) × (7.7-)7.7-8.5(-8.6) μm, (mean ± SD = 12.3 ± 0.6 × 8.1 ± 0.4 μm, n = 6).

Culture characteristics.

Colonies on PDA growing in the dark with an average growth rate of 9.2 mm/d at 25 °C. Colony color white, flat, margins regular and fimbriate. Aerial mycelia abundant. Odor absent. Reverse white to pale luteous. Colonies on SNA incubated at 25 °C in the dark growing at 7.2 mm/d. Colony surface pure white, aerial mycelium scant. Reverse pure white, without diffusible pigments. Colonies on OMA incubated at 25 °C in the dark growing at 10.1 mm/d, color white, flat, velvety to felty with abundant floccose aerial mycelium. Reverse white without diffusible pigments. Colonies on CMA incubated at 25 °C in the dark were round, colony surface and reverse white, flat, aerial mycelium absent, hyphae hyaline, growing at 9.1 mm/d.

Additional materials examined.

China, Hunan province, Yiyang City, Heshan District, Henglongqiao Town , 28°27′24″N, 112°29′7″E, isolated from leaf spots of Cunninghamia lanceolata , May 2017, Wen-Li Cui, isolates: HN33-8-2-1, HN33-8-2-2, HN33-8-2-3 .

Notes.

The isolates of F. hunanense were phylogenetically close to F. pseudensiforme (ex-type, CBS 125729) (Fig. 4 ). Between F. hunanense isolates and ex-type of F. pseudensiforme CBS 125729, there were 8/583 differences in TEF-1α, 3/800 in RPB2, and 9/640 in RPB1. The PHI analysis showed that there was no significant recombination among F. hunanense isolates and its related species (Φw = 1.0) (Fig. 3B ). Morphologically, 5-septate sporodochial macroconidia of the F. hunanense isolates (60.6-73.0 × 6.1-7.1 µm) were longer than those of F. pseudensiforme CBS 125729 (ex-type) (50-63 × 5.2-7.2 µm) ( Nalim et al. 2011). In conclusion, the phylogenetic and morphological evidence supported this fungus being a new species within the F. solani species complex.

Pathogenicity assays.

Pathogenicity was tested on detached C. lanceolata leaves in vitro following Koch’s postulates for F. hunanense (HN33-8-2), F. concentricum (SJ1-10), F. guizhouense (GZ7-20-1), F. fujikuroi (HN43-17-1), and F. fujianense (LC14). At five days post-inoculation, all the tested isolates caused leaf necrosis, with dark brown lesions. The control remained unchanged (Fig. 9A ). Equivalently, shoots of tissue-culture seedlings of C. lanceolata were inoculated by F. hunanense (HN33-8-2), F. concentricum (SJ1-10), F. guizhouense (GZ7-20-1), F. fujikuroi (HN43-17-1), and F. fujianense (LC14) in vivo. After ten days post-inoculation, all isolates caused necrotic lesions on shoots of C. lanceolata . The control remained healthy (Fig. 9B ). Statistically, these isolates showed different levels of virulence. Fusarium hunanense (HN33-8-2) was significantly more virulent than those of F. concentricum (SJ1-10), F. guizhouense (GZ7-20-1), F. fujikuroi (HN43-17-1), and F. fujianense (LC14), while F. fujianense (LC14) was the least virulent (Fig. 9C ).

The fungal isolates used for inoculation were re-isolated from the diseased spots on the inoculated leaves and shoots, but no fungus was isolated from the leaves and shoots of the control. Koch’s postulates were satisfied, and these isolates HN33-8-2, SJ1-10, GZ7-20-1, HN43-17-1, and LC14 were determined to be the pathogens of leaf blight on C. lanceolata .

Figures

Figure 3. Splitgraphs showing the results of the pairwise homoplasy index (PHI) test of three newly described taxa and closely related species using both LogDet transformation and splits decomposition A the PHI of Fusarium fujianense sp. nov. with their phylogenetically related isolates or species B the PHI of F. hunanense sp. nov. with their phylogenetically related isolates or species C the PHI of F. guizhouense sp. nov. with their phylogenetically related isolates or species. PHI test value (Φw) <0.05 indicate significant recombination within a dataset. * indicates isolates of this study. T indicates ex-types. HT indicates ex-holotypes.

Figure 4. Phylogenetic relationships of 41 isolates of the Fusarium solani species complex with related taxa with concatenated sequences of the TEF- 1 α, RPB 2, and RPB 1 loci using Bayesian inference (BI) and maximum likelihood (ML) methods. Bootstrap support values from ML ≥ 70 % and BI posterior values ≥ 0.9 are shown at nodes (ML / BI). Fusarium staphyleae NRRL 22316 and F. cicatricum CBS 125552 were the outgroup. * indicates strains of this study. T indicates the ex-type strains. ET indicates ex-epitypes.

Figure 8. Fusarium hunanense (HN 33 - 8 - 2) A-D colonies on PDA, SNA, OMA, and CMA, respectively, after 5 days at 24 ° C in the dark E sporodochia formed on PDA F-K aerial conidiophores, phialides, and conidia L-N sporodochial conidiophores, phialides, and conidia O, P macroconidia (3 - 6 - septate) Q chlamydospore. Scale bars: 1,000 μm (E); 50 μm (F-H); 10 μm (I-Q).

Figure 9. Symptoms on detached Cunninghamia lanceolata leaves (A) and shoots of tissue-culture seedlings of C. lanceolata (B) inoculated with isolates: Fusarium fujianense (LC 14), F. fujikuroi (HN 43 - 17 - 1), F. guizhouense (GZ 7 - 20 - 1), F. concentricum (SJ 1 - 10), and F. hunanense (HN 33 - 8 - 2). Scale bar: 10 mm. C, Lesion length on detached C. lanceolata leaves inoculated with F. fujianense (LC 14), F. fujikuroi (HN 43 - 17 - 1), F. guizhouense (GZ 7 - 20 - 1), F. concentricum (SJ 1 - 10), and F. hunanense (HN 33 - 8 - 2). Error bars represent standard deviation, and different letters indicate significant difference based on LSD's range test at P <0.05 (n = 8).