Sequence analysis
We estimated the pairwise similarity between the ITS sequence of the query strain and the sequences that are given in the ITS56 datasets. All the query strain was found to belong to the genus Trichoderma with similarity value > 83% compared to the sequences in the datasets. The query strain had unique sequences of tef1 and rpb2 (i.e., it did not meet the sp∃!(rpb299 ≅ tef197) standard for known Trichoderma species. The tef1 or rpb2 sequences of Trichoderma strain in this study were subjected to MMIT and NCBI nucleotide BLAST, and 104 representative strains of 71 closely related Trichoderma species were chosen for phylogenetic analyses. The best-fit models TPM3 + F + G4 T and Ne + G4 were selected using the BIC criterion for ML analyses of tef1 and rpb2, respectively, and the ML phylogenetic trees are shown in Figs. 1. Meanwhile, K2P + G4 and TNe + R3 were selected for tef1 and rpb2, respectively, and the ML phylogenetic trees are shown in Figs. 2.
Each putative new taxon of Trichoderma in this study formed a distinct clade and meeting the Trichoderma new species standard (i.e., did not meet the sp∃!(rpb299 ≅ tef197) for all known Trichoderma species (Cai and Druzhinina 2021).
T32450, T32781 and T32794 clearly separated from T. obobatum YMF 1.06211 and YMF 1.6190 with similarity of rpb2 = 98.15% and tef1 = 86.68%, T. uncinatum YMF 1.04622 with similarity of rpb2 = 98.56% and tef1 = 94.40%, T. paratroviride CBS 136489 and S489 with similarity of rpb2 = 98.65% and tef1 = 87.53%, T. atroviride CBS 142.95 and 119499 with similarity of rpb2 = 97.91 ~ 97.79% and tef1 = 91.29% (Fig. 1).
T31818, T32434, T32465 and T32353 clearly separated from T. pyramidale CBS 135574 and with similarity of rpb2 = 98.02% and tef1 = 93.40%, T31823 with similarity of rpb2 = 97.5% and tef1 = 99.2%, T. guizhouense strain CBS 131803 with similarity of rpb2 = 97.05% and tef1 = 98.29%, T. asiaticum YMF1.00352 with similarity of rpb2 = 96.92% and tef1 = 98.06%, and T. simile YMF1.06201 with similarity of rpb2 = 97.17% and tef1 = 97.83% (Fig. 2).
T31823 and T31824 clearly separated from T. zelobreve CGMCC 3.19695 with similarity of rpb2 = 97.54% and tef1 = 96.35%, T32434 with similarity of rpb2 = 97.5% and tef1 = 99.2%, T. guizhouense strain CBS 131803 with similarity of rpb2 = 97.17% and tef1 = 98.29%, T. asiaticum YMF1.00352 with similarity of rpb2 = 97.79% and tef1 = 98.06%, and T. simile YMF1.06201 with similarity of rpb2 = 97.66% and tef1 = 97.83% (Fig. 2).
Taxonomy
Trichoderma nigricans C.L. Zhang, sp. nov. Figure 3.
MycoBank
845506
Etymology
The Latin specific epithet “nigricans" refers to the “blackish green” color of the mass of conidia.
Diagnosis: Phylogenetically T. nigricans formed a distinct clade and is related to T. atroviride, T. paratroviride, T. obovatum and T. uncinatum (Fig. 1). The similarity of rpb2 found at this locus between strain T. nigricans and the most closely related species T. paratroviride (strain CBS 136489), T. uncinatum (strain YMF 1.6190 and YMF 1.06211) and T. obovatum (strain YMF 1.04622) was 98.65%, 98.65% and 98.15%, respectively. The similarity of tef1 found at this locus between strain T. nigricans and the most closely related species T. uncinatum (strain YMF 1.06211) was 94.40% (i.e., the condition sp∃!(rpb299 ≅ tef197) was not met). This indicates that T. nigricans could be recognized as new species. T. nigricans is characterized by its growth: colony radius on CMD after 72 h, mycelium covers the plate at 25 ℃ and 30 ℃; on PDA, mycelium covers the plate at 25 ℃, grows faster than T. atroviride, T. paratroviride, T. obovatum and T. uncinatum.
Type: CHINA: Shandong Province, Dezhou City, 37°21'07"N, 116°23'40"E, 5 m alt., isolated from soils of peach rhizosphere. Oct 2015, Y. Jiang T32781 (Holotype CGMCC40314, stored in a metabolically inactive state).
Description: Optimal growth at 25 ℃, slow at 35 ℃ on all media. Colony radius on CMD after 72 h: mycelium covers the plate at 25 ℃ and 30 ℃, 20–22 mm at 35 ℃. Colony well-defined, hyaline, sparse aerial mycelia, indistinctly zonate, conidiation begins to develop within 72 h, white at first and turning green after 3–4 d. After 7 d, abundant dark green conidiation around the margin, radially arranged within 2–3 ill-defined concentric zones in the outer half of the colony. Abundant chlamydospores. No diffusing pigment noted, pleasant odor apparent. Colony radius on PDA after 72 h: mycelium covers the plate at 25 ℃, 55–61 mm at 30 ℃, 16 mm at 35 ℃. Colony similar to CMD but growth a little slower, colony not dark green. Colony well-defined at 35 ℃, abundant white thick aerial mycelia. Chlamydospores abundant. No diffusing pigment noted, obvious pleasant odor. Colony radius on MEA after 72 h: 58–60 mm at 25 ℃, 53–55 mm at 30 ℃, 11–12 mm at 35 ℃. Colony also similar to CMD, but conidiation is yellow green, more abundant around the inoculation plug, uniform distribution all around. No diffusing pigment noted, odor indistinct. Colony radius on SNA after 72 h: 5–7 mm at 25 ℃, 5–6 mm at 30 ℃ and 35 ℃. Colonies well-defined, hyaline, scant aerial mycelia. Slight conidiation dispersedly distributed around the inoculation plug, with white floccose indistinctly zonate tufts or pustules in the margin. No diffusing pigment noted, odor indistinct. Conidiophores consisting of a main axis with side branches mostly at right angles or slightly inclined upward; branches straight or curved, often only longer in basal positions, not re-branching, solitary, paired or in whorls of three. Phialides solitary or commonly in whorls of 2–3, variable in shape, either narrowly lageniform to subulate, particularly when terminal on the main axis, or stout to nearly ampulliform and distinctly swollen, sometimes ampulliform to subglobose, (4.7-)6.0-8.9(-12.1) × (2.5-)2.9–3.4(-4.5) µm (mean = 7.7 × 3.3 µm), base 1.8–2.4 µm (mean = 2.2 µm); phialide length/width ratio (1.2-)1.8–2.9(-3.6) (mean = 2.4); Conidia subglobose to globose, green, smooth, (3.0-)3.2–3.6(-3.9) ×(2.8-)3.1–3.4(-3.8) µm (mean = 3.3×3.4 µm), with length/width ratio of 1.0-1.1 (mean = 1.1). Abundant chlamydospores, common single, sometimes terminal and intercalary, globose to subglobose, (7.2-)7.8–9.2(-10.1) × (6.1-)7.1-9.0(-9.7) µm (mean = 8.6×8.1 µm).
Sexual morph
Unknown.
Substrate
Soils.
Distribution
China, Shandong Provinces.
Additional material examined: CHINA: Shandong Province, Jinan City, 36°33'45"N, 116°57'05"E, 105 m alt., isolated from corn soils. Aug 2015, Y. Jiang T32450. CHINA: Shandong Province, Dezhou City, 37°21'07"N, 116°23'40"E, 5 m alt., isolated from soils of corn rhizosphere, Oct 2015, Y. Jiang, T32794.
Notes
T. nigricans can be distinguished from similar species based on growth. After 72 h at 25 ℃, T. nigricans mycelium covers the plate on PDA and CMD, T. atroviride grows to 42.8–60.5 mm on PDA, T. obovatum grows to 38–41 mm on CMD, T. uncinatum grows to 55-62mm on CMD, T. paratroviride to 49–62 mm on CMD and 54–56 mm on PDA (Bissett 1992; Jaklitsch and Voglmayr 2015; Samuels et al. 2002; Zheng et al. 2021). In addition, it can distinguished by its chlamydospores and odor. At 35 ℃ the growth of T. nigricans is restricted, and no growth occurs in T. paratroviride and T. uncinatum. The chlamydospores are unobserved in T. obovatum and T. uncinatum, and uncommon of T. paratroviride. Meanwhile, the chlamydospores of T. atroviride and T. nigricans are abundant, and the volume in T. atroviride is usually larger than those in T. nigricans [(5.2-)8.5–12.0(-16.3) vs. (7.2-)7.8–9.2(-10.1) × (6.1-)7.1-9.0(-9.7) µm]. On PDA, the odor of T. paratroviride is pungent, it is indistinct in T. obovatum and T. uncinatum, and pleasant in T. atroviride and T. nigricans.
Trichoderma densissimum C.L. Zhang, sp. nov. Figure 4.
MycoBank
845507
Etymology
The Latin specific epithet “densissimum” refers to the thick wall of chlamydospores of this species.
Diagnosis
The similarity of rpb2 between strain T. densissimum and the most closely related species T. pyramidale (strain CBS 135574) that is found at this locus was 98.02%, while the similarity of tef1 found at this locus between strain T. densissimum and the most closely related species including T. paradensissimum (strain T31823 and T31824), T. guizhouense (strain CBS 131803), T. asiaticum (strain YMF1.00352) and T. simile (YMF1.06201) was 99.2%, 98.29%, 98.06% and 97.83%, respectively (i.e., the condition sp∃!(rpb299 ≅ tef197) was not met). This indicates that strain T. densissimum can be recognized as new species. It is easily distinguished from these related species by its relatively large chlamydospores (11.7-)13.3–16.4 (-19.5) × (11.5-)12.8-14.6-12.8 (-16.0) µm (mean = 14.8 × 13.6 µm).
Type: CHINA: Shandong Province, Weifang City, 36°38'27"N, 119°01'21"E, 80 m alt., isolated from soils of apple tree rhizosphere. Oct 2015, Y. Jiang T32434 (Holotype CGMCC3.24126, stored in a metabolically inactive state).
Description: Optimum temperature for growth is 30 ℃ on CMD, MEA and SNA and 25 ℃ on PDA. Growth slow at 35 ℃ on PDA and SNA. Chlamydospores are common on all media. Colony radius on CMD after 72 h: 38–45 mm at 25 ℃, 55–62 mm at 30 ℃, 42–43 mm at 35 ℃. Colonies well-defined, white, thin, aerial hyphae sparse. Conidiation was noted after 2 d around the inoculation plug, which was white at first, turning yellow green after 3–4 d, then dark green after 5–6 d. Conidiation formed 4 obvious concentric zones. No diffusing pigment noted, odor indistinct. Chlamydospores common single, sometimes terminal and intercalary, globose to subglobose, (11.7-)13.3–16.4(-19.5) × (11.5-)12.8-14.6-12.8(-16.0) µm (mean = 14.8 × 13.6µm); with length/width ratio of 1.0 × 1.3 (mean = 1.1). Colony radius on PDA after 72 h: 61–66 mm at 25 ℃, 60–63 mm at 30 ℃, 24–31 mm at 35 ℃. Colony white, regularly circular, distinctly zonate; mycelium dense and radial. Conidiation in the form on pustules, yellow-green, relatively abundant in the zonation regions. No diffusing pigment noted, odor indistinct. Colony radius on MEA after 72 h: 62–63 mm at 25 ℃, 66–67 mm at 30 ℃, 44–47 mm at 35 ℃. Colonies similar to that on PDA, but indistinctly zonate. No diffusing pigment noted, odor indistinct. Colony radius on SNA after 72 h: 53 mm at 25 ℃, 41–47 mm at 30 ℃, 27–32 mm at 35 ℃. Colony white; aerial mycelia scant and loose. Conidiation in the form of minute pustules, radial and inconspicuously zonate. No diffusing pigment noted, odor indistinct. Conidiophores pyramidal with opposing branches, the main axis with side branches is sometimes at right angles or inclined upward. The main axis and each branch commonly terminating verticillate, whorl of 3–4 phialides, sometimes in a cruciate whorl, sometimes solitary phialides. Phialides commonly ampulliform, sometimes ampulliform to subglobose (3.4-)5.7-8.0(-10.1) × (1.9-)2.5–2.9(-3.2) µm (mean = 6.2 × 2.6µm), base (1.0-)1.4–2.1(-2.6) µm (mean = 2.2 µm); phialide length/width ratio (1.4-)2.1–3.2(-3.9) (mean = 2.6); Conidia subglobose to globose, green, (2.3-)2.8–3.1(-3.4) × (2.2-)2.4–2.9(-3.3) µm (mean = 2.9 × 2.7 µm), with length/width ratio of 1.0-1.4 (mean = 1.1).
Sexual morph
Unknown.
Substrate
Soils.
Distribution
China, Shandong and Shanxi provinces.
Additional material examined: CHINA: Shandong Province, Jinan City, 36°32'33"N, 117°01'08"E, 201 m alt., isolated from soils of wheat, Jun 2015, Y. Jiang (T31818); Shandong Province, Jining city, 34°56'21"N, 116°29'03"E, 34 m alt., isolated from soils of peach, Aug 2015, Y. Jiang T32353; Shanxi Province, Baoji city, 34°23'25"N, 107°10'18"E, 802 m alt., isolated from soils of corn, Aug 2015, Y. Jiang T32465.
Notes
Although T. densissimum, T. paradensissimum and T. guizhouense share similar conidia and pyramidal conidiophores, but T. densissimum cannot produce pigments while T. paradensissimum can produce yellowish pigment on PDA and CMD at 35℃ in the dark (Li et al. 2013). T. guizhouense chlamydospores unobserved. Different from T. pyramidale, it can grow at 35 ℃ and chlamydospores are common. Characterized by producing globose to subglobose chlamydospores, the chlamydospores of T. simile are elliptic or round and unobserved in T. asiaticum (Jaklitsch and Voglmayr 2015; Zheng et al. 2021).
Trichoderma paradensissimum C.L. Zhang, sp. nov. Figure 5.
MycoBank
845508
Etymology
The Latin specific epithet “para” means similar, and “paradensissimum” refers to the phylogenetic proximity and morphological similarity to T. densissimum.
Diagnosis
The sequence similarity of rpb2 between strain T. paradensissimum and the most closely related species T. zelobreve CGMCC 3.19695 was 98.77%. The sequence similarity of tef1 with T. densissimum, T. guizhouense (strain CBS 131803), T. asiaticum (strain YMF1.00352) and T. simile (YMF1.06201) was 99.2%, 98.29%, 98.06% and 97.83%, respectively (i.e., the condition sp∃!(rpb299 ≅ tef197) was not met). This indicates that T. paradensissimum can be recognized as new species. T. paradensissimum is characterized by the green to yellow and white pustules formed inconspicuously zonate on PDA or MEA at 25 ℃ of a 12- h photoperiod after 7 d.
Type: CHINA: Shanxi Province, Jincheng City, 35°26'57.9"N, 112°45'19.0"E, 929 m alt., isolated from soils of wheat rhizosphere, Jun 2015, Y. Jiang T31823 (Holotype CGMCC3.24125, stored in a metabolically inactive state).
Description. Optimum temperature for growth is 30 ℃ on CMD, PDA and SNA and 25 ℃ on MEA. Chlamydospores were common on all media. Colony radius on CMD after 72 h: 40–42 mm at 25 ℃, 63–64 mm at 30 ℃, 38–40 mm at 35 ℃. Colony well-defined, white, aerial myceli loose and radial. White minute pustules were noted after 2 d around the inoculation plug, white at first, turning yellow green after 3–4 d, then dark green after 5–6 d. Around the point of inoculation, conidiation from dark green to pale green, inconspicuously zonate. Distinctive odor absent. The production of pigment was related to light, media and temperature: around the point of inoculation, it was yellowish at 35 ℃ in the dark. Colony radius on PDA after 72 h: 59–65 mm at 25 ℃, 64–67 mm at 30 ℃, 20–24 mm at 35 ℃. Colonies similar to that on CMD. Pustules were noted after 4–5 d. After 7 d, the green to yellow and white pustules were formed as inconspicuously zonate. Distinctive odor absent. The production of pigment was related to light and temperature, it was yellowish at 35℃ in dark. Colony radius on MEA after 72 h 58–59 mm at 25 ℃, 51–53 mm at 30 ℃, 34–35 mm at 35 ℃. Colonies white and thick, regularly circular and radial, aerial myceli dense. A few white-yellow large pustules formed inconspicuously zonate. Diffusing pigment or distinctive odor absent. Colony radius on SNA after 72 h 35–37 mm at 25 ℃, 43–44 mm at 30 ℃, 15–16 mm at 35 ℃. Colony pale white; aerial myceli loose. Conidiation was minute pustules, radial and inconspicuously zonate. Around the point of inoculation, the pustules were green, but white far away from the inoculation. Diffusing pigment or distinctive odor absent. Conidiophores pyramidal; the main axis with side branches sometimes at right angles or inclined upward. The main axis and each branch commonly terminating verticillate, whorl of 3 phialides, sometimes solitary. Phialides ampulliform, (5.4-)7.4–11.0(-15.0) × (2.1-)2.7–3.1(-3.3) µm (mean = 9.4 × 2.9 µm), base (1.6-)1.8–2.3(-2.6) µm (mean = 2.0 µm); phialide length/width ratio (2.1-)2.6–3.7(-4.9) (mean = 3.2); Conidia subglobose to globose, green, (2.6-)2.7-3.0(-3.5) × (2.4-)2.5–2.9(-3.2) µm (mean = 2.9 × 2.7 µm), with length/width ratio of 1.0-1.2 (mean = 1.1). Chlamydospores abundant, common single, sometimes terminal and intercalary, globose to subglobose, (4.6-)5.1–6.2(-6.8) × (3.7-)4.6–5.9(-6.7) µm (mean = 5.7 × 5.4 µm); length/width ratio 1.0×1.3(mean = 1.1).
Sexual morph
Unknown.
Substrate
Soils.
Distribution
China, Shanxi Province.
Additional material examined: CHINA: Shanxi Province, Jincheng City, 35°26'58.1"N, 112°45'19.4"E, 929 m alt., isolated from soil of wheat rhizosphere, Jun 2015, Y. Jiang T31824.
Notes: Similar species can be distinguished according to the pigment:, T. paradensissimum can produce yellowish pigment on PDA and CMD at 35℃ in the dark, whereas T. guizhouense typically at 35 ℃ reverse forming a dull orange to brown pigment. However, T. densissimum, T. asiaticum, T. simile and T. zelobreve cannot produce diffusing pigment on PDA.