Trichoderma: Identification and Agricultural Applications

TRICHODERMA Identification and Agricultural Applications Gary J. Samuels and Prakash K. Hebbar

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Trichoderma Species Descriptions

In the following descriptions, growth rates are reported as the colony diameter after incubation in darkness (with intermittent light when cultures were measured).

The radius is measured from the edge of the 3- to 5-mm plug of inoculum to the most distant point of the colony. The colony radius is given in millimeters (mm). The description of conidiation and the observations of conidia

and phialides are taken from colonies grown on Spezieller Nährstoffarmer agar (SNA) for 1 week at 25°C under a 12-h darkness/12-h cool white fluorescent light regime.

Chlamydospores are observed at 20× by scanning a colony in an inverted 9-cm-diameter petri plate using brightfield microscopy. Illustrations of colonies grown in petri plates are shown in Figures 6.1–6.4.

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Figure 6.1. Cultures of Trichoderma species. Except where noted, cultures were grown for 1 week at 25°C under cool white fluorescent light; potato dextrose agar (PDA) on the left, Spezieller Nährstoffarmer agar (SNA) on the right. A, T. aggressivum.

B, T. amazonicum. C, T. asperellum. D, T. atroviride. E, T. aureoviride, crystals in malt extract agar. F, T. brevicompactum.

G, T. capillare. H, T. citrinoviride. I, T. crassum. J, T. deliquescens on PDA. K, T. effusum. L, T. erinaceus was grown for 1 week at 25°C in the dark.

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Figure 6.2. Cultures of Trichoderma species. Cultures were grown at 25°C under cool white fluorescent light; potato dextrose agar on the left, Spezieller Nährstoffarmer agar (SNA) on the right. A, T. evansii. B, T. flagellatum. C, T. gamsii. D, T. ghanense.

E, T. hamatum. F, T. harzianum. G, T. koningii. H, T. koningiopsis. I, T. lieckfeldtiae. J, T. longibrachiatum. K, T. longipile.

L, T. minutisporum. A–C and J–L, Cultures were grown for 1 week. D and F–H, Cultures were grown for 96 h.

E and I, Cultures were grown for 10 days.

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Figure 6.3. Cultures of Trichoderma species. Cultures were grown at 25°C under cool white fluorescent light; potato dextrose agar (PDA) on the left, Spezieller Nährstoffarmer agar (SNA) on the right. A, T. orientale (right is PDA reverse to show diffusing yellow pigment). B, T. ovalisporum. C, T. paucisporum, a conidial pustule on PDA is shown on the right. D, T. pleuroti.

E, T. pleuroticola. F, T. polysporum. G, T. pseudokoningii. H, T. pubescens. I, T. reesei. J, T. rossicum. K, T. saturnisporopsis.

L, T. saturnisporum. A, G, I, and K, Cultures were grown for 1 week. B, C, and L, Cultures were grown for 96 h.

D, E, and J, Cultures were grown for 10 days. F and H, Cultures were grown for 2 weeks.

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Figure 6.1

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Trichoderma Species Descriptions

Figure 6.2

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Figure 6.3

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Trichoderma Species Descriptions

Figure 6.4. Cultures of Trichoderma species. Cultures were grown at 25°C under cool white fluorescent light (except where noted); potato dextrose agar on the left, Spezieller Nährstoffarmer agar (SNA) on the right. A, T. spirale. B, T. stilbohypoxyli.

C, T. strigosum. D, T. stromaticum. E, T. tomentosum. F, T. velutinum, grown in darkness. G, T. virens. H, T. viride.

I, T. viridescens. A, C–E, G, and I, Cultures were grown for 1 week. B, F, H, Cultures were grown for 96 h.

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Trichoderma aggressivum ●

Samuels & W. Gams in Dodd et al., Mycologia 94:167. 2002. Figs. 6.1A, 6.5.

Ex-type culture: DAOM 222156 = IMI 393971 GenBank: rpb2: AF545541; tef1: AF348098

Clade: Harzianum Colony radius at 25°C: PDA (33–)45–60. SNA (42–) 55–65(–68). No diffusing pigment or distinctive odor on PDA. Colony radius at 35°C: PDA (1–)3–10(–12).

SNA (4–)8–25(–32).

Conidiation: Pustules scattered throughout the colony, 1–2 mm in diameter, loosely cottony, grayish green; conidia appearing dry.

Microscopic characteristics: Branching pattern typically Harzianum type with phialides held in divergent whorls on frequently paired or verticillately whorled branches; solitary phialides and unilateral branching and extended conidiophores common. Sterile hairs not formed. Phialides ampulliform, sharply constricted below the tip to form a narrow neck and slightly constricted at the base, (4.0–)5.7–7.7(–21.0) µm long, (1.3–)2.7–3.5(–4.3) µm wide at the widest point, L/W (1.2–)1.7–2.3(–8.0). Supporting cell (1.5–)2.5– 3.7(–5.2) µm wide; ratio of length of phialide to width of supporting cell (1.0–)1.7–3.2(–8.0); ratio of width of phialide to width of supporting cell 1. Conidia subglobose to ovoidal, (2.0–)2.5–3.2(–3.7) × (2.0–)3.0– 3.5(–5.5) µm, L/W (0.7–)1.0–1.3(–1.7), smooth, green.

Chlamydospores few.

Distinguishing characters: Trichoderma aggressivum is most similar to T. harzianum from which it differs in its frequently inequilaterally branched conidiophores and generally very slow rate of growth at 35°C. Habitat: Mushroom compost, causing green mold disease of commercial mushrooms (Agaricus bisporus).

Not found apart from mushroom compost. On Pleurotus ostreatus, compare with T. pleuroti and T. pleuroticola.

Distribution: North America, Europe.

Literature: Samuels et al. (2002), Seaby (1998; as T. harzianum Group IV).

Figure 6.5. Trichoderma aggressivum. A–H, Conidiophores. I, Conidia. A, B, E, F, H, and I, From cornmeal dextrose agar.

C, D, and G, From Spezieller Nährstoffarmer agar (SNA). Scale bars: A–G = 20 µm; H, I = 10 µm.

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Trichoderma amazonicum ●

P. Chaverri & Gazis in Chaverri et al., Mycologia 103:146. 2011. Figs. 6.1B, 6.6.

Ex-type culture: CBS 126898 GenBank: rpb2: HM142368; tef1: HM142376

Clade: Harzianum Colony radius at 25°C: PDA 55–60. SNA 45–50.

No diffusing pigment or distinctive odor on PDA or a slightly fruity odor and/or brownish diffusing pigment observed in some cultures.

Colony radius at 35°C: PDA 0 or 40–55. SNA 0 or 35–45.

Conidiation: Discrete pustules not forming or, at most, conidiophores loosely aggregated into ill-defined pustules in the colony center, typically conidia forming in broad, more-or-less continuous, cottony concentric rings, green; conidia appearing dry.

Microscopic characteristics: Branching pattern Pachybasium type with a well-defined, conspicuously broad (3–5 µm wide) main axis and broad primary branches arising along the length in whorls of two or three, increasing in length with distance from the tip, rebranching or producing whorls of phialides directly; secondary branches one or a few cells in length, terminating in three to several phialides. Phialides ampulliform, typically sharply constricted to a narrow neck, 4.5–6.5 µm long, 2.7–3.7 µm at the widest point, L/W 1.5–2.2. Supporting cell 3–4 µm wide; ratio of length of phialide to width of supporting cell 1.4–2.0; ratio of width of phialide to width of supporting cell approximately 1. Conidia broadly ellipsoidal to subglobose, (1.7–)2.2–2.7(–3.0) × (2.0–)2.5–3.2(–3.5) µm, L/W (0.8–)1.0–1.4(–1.6), smooth, green. Discrete chlamydospores not observed but tight clusters of globose to subglobose chlamydospore-like structures observed after 3 weeks on PDA; these structures are capable of germinating.

Distinguishing characters: The conidiophores with a strongly developed and conspicuously broad main axis and uniformly spaced whorls of branches are characteristic. The closest morphological comparison is with T. pleuroticola, but conidia of T. pleuroticola are formed in more-or-less discrete pustules. Chaverri et al. (2011) reported no growth for endophytic strains of T. amazonicum at 35°C and somewhat larger conidia; the description given here is based on strains isolated from soil in Peru, which did grow at 35°C. Habitat: Endophytic in sapwood and leaves of Hevea species and in soil.

Distribution: Common in Amazonian Peru, not known from elsewhere.

Literature: Chaverri et al. (2011).

Figure 6.6. Trichoderma amazonicum. A and B, Pustules of conidia. C–H, Conidiophores. I and J, Phialides. K, Conidia.

A–K, From Spezieller Nährstoffarmer agar (SNA). Scale bars: A = 1 mm; B = 0.5 mm; C–E, G, H = 20 µm; F, I–K = 10 µm.

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Trichoderma asperellum ●

Samuels, Lieckf. & Nirenberg in Samuels et al., Sydowia 51:81. 1999. Figs. 6.1C, 6.7.

Ex-type culture: CBS 433.97 GenBank: rpb2: EU248617; tef1: AF456907, AY376058

Clade: Viride Colony radius at 25°C: PDA (10–)37–57(–60).

SNA (10–)28–46(–48). No pigment or distinctive odor on PDA. Colony radius at 35°C: PDA (2–)17–37(–45).

SNA (1–)13–37(–43).

Conidiation: Pustules forming abundantly in two or more often conspicuous concentric rings, 1–2 mm in diameter, hemispherical, loosely organized with conidiophores visible with stereomicroscope, dark green; conidia appearing dry.

Microscopic characteristics: Branching pattern Pyramidal type with verticillate or frequently conspicuously paired lateral branches arising from a well-developed main axis; lateral branches rebranching; all branches arising at approximately 90° with respect to the subtending branch. Sterile hairs not formed. Phialides terminating branches, held in divergent whorls of two to four, lageniform, (4.0–)6.2–9.7(–15.2) µm long, (2.5–)3.0–3.5(–5.0) µm at the widest point, L/W (1.3–)1.7–3.1(–5.4). Supporting cell (1.7–)2.5–3.5(–5.7) µm wide; ratio of length of phialide to width of supporting cell (1.7–)2.0–3.4(–4.8); ratio of width of phialide to width of supporting cell (0.6–)0.9–1.3(–1.9). Conidia subglobose to ovoidal, (2.2–)3.0–3.5(–4.7) × (3.0–)3.5–4.0(–4.7) µm, L/W (0.9–)1.0–1.3(–1.7), green, finely warted (often appearing smooth). Chlamydospores not observed in all cultures, subglobose, terminal.

Distinguishing characters: Rather large, subglobose to ovoidal, finely ornamented conidia are produced from slender phialides on long, very uniformly branched conidiophores in dark green pustules; fast rate of growth at 35°C. The conidia often appear smooth. Trichoderma asperellum is morphologically indistinguishable from T. asperelloides (Samuels et al., 2010).

Habitat: Soil.

Distribution: Cosmopolitan.

Literature: Chutrakul et al. (2008; trichotoxin, peptaibols), Lieckfeldt et al. (1999), Samuels et al.

(1999, 2010).

Figure 6.7. Trichoderma asperellum. A–C, Conidial pustules. Note the projecting conidiophores in B and C. D–G, Conidiophores.

H and I, Conidia (optical section in H; surface view in I). A and H–I, From cornmeal agar. B and D–G, From cornmeal dextrose agar. C, From Spezieller Nährstoffarmer agar (SNA). Scale bars: A, B = 1 mm; C = 0.5 mm; D–G = 20 µm; H, I = 10 µm.

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