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Trichoderma stromaticum sp. nov., a parasite of the cacao witches broom pathogen
760
Mycol. Res. 104 (6) : 760–764 (June 2000). Printed in the United Kingdom.
Trichoderma stromaticum sp. nov., a parasite of the cacao witches broom pathogen
G. J. SAMUELS1*, R. PARDO-SCHULTHEISS1, K. P. HEBBAR2, R. D. LUMSDEN2, C. N. BASTOS3, J. C. COSTA4 and J. L. BEZERRA4 " United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Laboratory, Room 304, B-011A, BARC-W, Beltsville, MD 20705, USA. # United States Department of Agriculture, Agricultural Research Service, Biocontrol of Plant Diseases Laboratory, B-011A, BARC-W, Beltsville, MD 20705, USA. $ CEPLAC\SUPOR, Rodovia Augusto Montenegro, Km 7, Belem-Para, CEP 66635-110, Brazil. % CEPLAC\CEPEC – Secag o de Fitopatologia, C. Postal 7, Itabuna, Bahia, CEP 45600-000, Brazil. E-mail : gary!fungi.ars-grin.gov Received 2 February 1999 ; accepted 20 August 1999.
A new species, Trichoderma stromaticum, was isolated from ‘ witches’ broom ’ of cacao (Theobroma cacao) in Brazil caused by Crinipellis perniciosa. The Trichoderma was reported earlier as T. viride and T. polysporum. Trichoderma stromaticum reduced new inoculum through the suppression of basidioma formation. It is characterised by having conidia that slowly become yellow-green in agar culture and by stout, ‘ Pachybasium-like ’, phialides formed on the surface of stromatic structures. The closest relations of T. stromaticum are T. harzianum and T. virens.
INTRODUCTION ‘ Witches’ broom ’ of cacao (Theobroma cacao), caused by the basidiomycete Crinipellis perniciosa, is one of the major problems of cacao culture in tropical South America and the Caribbean islands (Bastos 1988). Symptoms of this disease include the formation of numerous small branches, termed brooms, from the infected meristems and flower cushions. There is no effective means of controlling the disease, although phytosanitation by removal of inoculum that forms from infected tissue in the brooms limits spread of the disease. Chemical controls have not proven effective but within the last ten years Bastos, Costa and their collaborators (Bastos 1988, 1996 a, b, c, Costa & Bezerra 1994, Costa, Bezzera & Cazorla 1996) have isolated a species of Trichoderma from dry brooms of cacao collected in Belem, Brazil. When applied to dry infected brooms, the Trichoderma prevented the formation of new inoculum through the suppression of basidioma formation. Moreover, no mycelium of the pathogen grew out of naturally infected brooms that were inoculated with conidia of the Trichoderma and then incubated in a damp chamber ; it was concluded that the Trichoderma was a direct parasite on mycelium of the pathogen (Bastos 1996 a). This Trichoderma has been misidentified and reported in the literature as T. viride (Bastos 1988, 1996 a, b, c, Costa & Bezerra 1994, and T. polysporum (Costa et al. 1996). Critical examination of the reported strains, however, reveals them to
be of an undescribed species of Trichoderma. Morphologically, it differs from T. viride (see Webster 1964, Lieckfeldt, Samuels & Nirenberg 1999, Samuels, Lieckfeldt & Nirenberg 1999) most conspicuously in forming smooth conidia from discrete, almost stromatic structures. Furthermore, the pattern of branching of conidiophores of this species is very different from what is found in T. viride. There is some similarity to T. polysporum (see Bissett 1991), in that both produce their conidia on discrete, cushion-shaped pustules. The similarity is only superficial, however, since conidia of T. polysporum are white to cream-coloured, only becoming at most pale green or grey with age, whereas conidia of the isolates from cacao are white for a long time but ultimately become green or yellow. Moreover, the pustules of T. polysporum are formed of hyphal elements that are relatively easily teased apart, while the pustules of the cacao isolates are very compact and formed of pseudoparenchymatous cells with conidia arising from conidiophores at the surface. The pustules of the cacao isolates are very compact and are only loosely attached to the agar surface ; they are easily dislodged en masse when the edge of the petri dish is tapped and themselves could possibly act as inoculum. The shape and arrangement of phialides indicate that the cacao isolates belong to Trichoderma sect. Pachybasium (Bissett 1991) but differ considerably from all species that were included in that section. It is, therefore, described below as T. stromaticum. Kindermann et al. (1998) found that Trichoderma
G. J. Samuels and others
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sect. Pachybasium is paraphyletic with respect to the ITS-1 region of rDNA and that some species that have a typical Pachybasium-like arrangement of phialides are more closely related to T. viride and other more typical species of Trichoderma than they are to each other. Thus, the morphology of T. stromaticum is not sufficient to predict its relationships. In an effort to determine the relationships of this fungus, analysis was undertaken of the ITS rDNA sequences in comparison with other species of Trichoderma.
above. Growth rates on potato dextrose agar (PDA, Difco) and ‘ special nutrient agar ’ (SNA, Nirenberg 1976) were determined following the scheme of Lieckfeldt et al. (1998). Colours are taken from Kornerup & Wanscher (1978). Sequences and fungal strains Sequences used in this study were obtained through GenBank. The isolates from cacao, GJS 97-179–97-183, from Brazil, are listed in Table 1.
MATERIALS AND METHODS Morphological observations
Nucleic acid extraction
All morphological observations are taken from cultures grown on cornmeal dextrose agar (CMD, Difco cornmeal agarj2 % glucose) at 20–21 mC, alternating cool white fluorescent light and dark. Conidial pustules were placed in a drop of 3 % KOH on a microscope slide and gently teased apart with needles. A cover slip was applied and observations were made with a light microscope. Water was added as necessary to prevent mounts from drying. Thirty of each morphological parameter were measured. Measurements are reported as the maximum and minimum of the means of the five collections of T. stromaticum.
Isolates of Trichoderma virens and T. stromaticum GJS 97-179–97-183 were grown on CMD plates at 20m for approximately one week. At this time, a 5 mm diam. agar plug was removed from the edge of each colony and placed in 15 ml liquid CYM (Raper & Raper 1972). Liquid cultures were maintained in darkness at room temperature for approximately 5 d, at which time the mycelial mat was transferred to sterile filter paper, blotted dry and frozen at k80m followed by lyophilization. The lyophilized mycelium was ground with a sterile applicator stick using liquid nitrogen in 1n5 ml microcentrifuge tubes. DNA extraction was performed following Rehner & Uecker (1994) and Rehner & Samuels (1994, 1995).
Cultural observations Morphology of colonies and conidial pustules was described from cultures grown on CMD under the conditions noted Table 1. Strains of Hypocrea and Trichoderma used and their GenBank accession numbers.
H. aureoviridis H. pachybasioides H. pilulifera T. atroviride T. hamatum T. hamatum T. harzianum T. inhamatum T. inhamatum T. koningii T. polysporum T. stromaticum T. stromaticum T. stromaticum T. stromaticum T. stromaticum T. virens T. virens T. viride T. viride
Strain
GenBank no.
CBS" 245.63 ATCC# 18650 CBS 814.68 DAOM$ 165779 ATCC 28012 DAOM 167057 CBS 819.68 CBS 273.78 CBS 518.81 CBS 459.96 CBS 820.68 GJS% 97-179 GJS 97-183 GJS 97-181 GJS 97-182 GJS 97-180 GL& 20 GLi& 39 CBS 240.63 GJS 89-127
Z48819 AF011929 Z48813 Z48817 X93975 Z48816 Z68189 Z68187 Z68188 Z79628 Z48815 AF098287' AF097913' AF097910' AF097912' AF097911' AF099007' AF099005' X93979 X93980
" CBS : Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. # ATCC : American Type Culture Collection, Manassas, VA, USA. $ DAOM : Eastern Cereals and Oilseeds Research Centre, Agriculture and
Agri-Food Canada, Ottawa, Ontario, Canada. % GJS : Collection of G. J. Samuels, Systematic Botany and Mycology Laboratory, US Dept of Agriculture, Beltsville, MD, USA. & GL, GLi : Biocontrol of Plant Diseases Laboratory, US Dept of Agriculture, Beltsville, MD, USA. ' Newly deposited sequences.
PCR and DNA sequencing PCR was used to amplify the ITS region of T. virens and the four cacao isolates. Amplification was performed in a 50 µl reaction volume using 5 µm of template DNA, 10i PCR buffer (Perkin Elmer\ABI, Foster City, CA), 200 m total dNTPs, 3 m MgCl , 1n25 Units Amplitaq Gold4 (Perkin # Elmer\ABI) and 25 pmole each of primers ITS5 and ITS4 (White et al. 1990). The PCR reaction mix was placed in a 0n2 ml microcentrifuge tube, the PCR carried out on a GeneAmp2 9700 PCR System (Perkin Elmer\ABI) and the samples amplified according to the following parameters : 10 min initial activation of Amplitaq Gold2 at 95m, denaturation for 30 s at 93m, annealing for 30 s at 55m, extension for 2 min at 72m for 35 cycles and a 10 min final extension period at 72m. Following amplification, residual nucleotides and primers were removed using the QIAquick4 PCR purification kit (QIAGEN Inc.) according to the manufacturer’s instructions and the PCR purified product eluted in QIAGEN elution buffer (10 m Tris.Cl, pH 8n5). The PCR templates were sequenced using the dRhodamine terminator cycle sequencing kit (Perkin Elmer\ABI) according to the manufacturer’s protocol. Sequenced product purification of unicorporated dye terminators from the extension products were removed with 70 % ethanol and 0n5 m MgCl (1000 : 1, # v\v) according to the manufacturer’s instructions. Extension products were eluted in 15 µl of template suppression reagent (TSR4) (Perkin Elmer\ABI) and analyzed on a model 310 fluorescent automated sequencer (Perkin Elmer\ABI). Sequences were determined on both strands using the following sequencing primers : ITS5, ITS4, ITS2 internal reverse and ITS3 internal forward (White et al. 1990).
Trichoderma stromaticum, sp. nov.
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Sequencing and phylogenetic analysis Sequences were edited, joined and aligned using the EditSeq, SeqMan II, and MegAlign modules in the Lasergene sequence software package (DNAStar, Inc.). Phylogenetic relationships were estimated from the aligned sequences and GenBank sequences using PAUP 3;1;1 (Swofford 1991). Maximum parsimony trees were inferred using the heuristic search (random sequence addition) and branch swapping (tree bisection-reconnection) options. All molecular characters were unordered and given equal weight during the analysis. Support for the phylogenetic groupings was obtained with bootstrap analyses (Felsenstein 1985) using 1000 replications with random addition input orders of sequences during each heuristic search. RESULTS The ITS rDNA data set for this study consisted of 17 taxa and approximately 640 bp per taxon, representing a total of 685 characters. For this data set, 76 characters were phylogenetically informative. Of these 76 characters, 45 were in the ITS1 region and 26 were in the ITS2 region. One informative character was in the beginning of the 5h end of the sequence that incorporates approximately 49 nucleotides of the 18S rDNA gene. Parsimony analysis produced four most parsimonious cladograms (Fig. 1) of 117 steps with a consistency index (CI) of 0n812 and a retention index (RI) of 0n925. T. virens (GL-20)
81
T. virens (GLi-39-type) 96
T. harzianum (CBS 819.68) 94
T. inhamatum (CBS 273.78)
76 72
100
T. inhamatum (CBS 518.81)
T. stromaticum (GJS 97-179) T. stromaticum (GJS 97-183-type)
H. aureoviridis (CBS 245.63) H. pilulifera (CBS 814.68) 95 100
H. pachybasioides (ATCC 18650) T. polysporum (CBS 820.68)
T. hamatum (ATCC 28012) H. rufa (GJS 89-127)
61 81 100 100
T. viride (CBS 240.63) T. koningii (CBS 459.96) T. atroviride (DAOM 165779)
T. hamatum (DAOM 167057 ex NT)
Fig. 1. Cladogram of phylogenetic placement of Trichoderma stomaticum. One of four most parsimonious trees (length l 117, CI l 0n812, RI l 0n925) inferred using heuristic and branch swapping options in Paup 3;1;1. All characters were unordered and given equal weight during analysis. Bootstrap support of 1000 replications is listed above the branches.
Bootstrap supports of 1000 replicates are indicated above the branches. The ITS sequence data for the five strains of T. stromaticum revealed that GJS 97-180 to GJS 97-183 had identical sequences while GJS 97-179 had two deletions (nt TT) at positions 206 and 207 and one substitution (nt C) at position 495. These deletions and substitutions were not phylogenetically informative, and all strains were identified as T. stromaticum. Analysis of the ITS region of this data set revealed that T. stromaticum is a terminal sister group to T. virens, T. harzianum and T. inhamatum. The strict consensus of the ITS region revealed high bootstrap values for most of the nodes in the cladogram. Hypocrea aureoviridis split the biocontrol group from the rest of the taxa examined, although this is only a reflection of the uncertainty of the placement of H. aureoviridis in relation to sect. Pachybasium. Groupings were consistent with data expressed in Kuhls et al. (1997) regarding the paraphyly of sect. Pachybasium. These data, however, only attempt to show the placement of T. stromaticum in relation to other Trichoderma species. From the molecular data, T. stromaticum represents a sister taxon to T. harzianum and T. virens. Trichoderma stromaticum Samuels & Pardo-Schultheiss, sp. nov. (Figs 2–13) Lente crescens. Conidia e pustulis 1–3 mm diam, pulvinatis vel subglobularibus exorientia. Cellulae pustularum vesiculares vel pseudoparenchymaticae, tenuitunicatae. Phialides dense dispositae, ex ramulis brevibus vel basim versus ex extensionibus subulatis conidiophororum oriundae, subglobosae vel ampulliformes, 5n5– 7n0 µm longae, 3n5–4n0 µm latae in medio. Conidia oblonga vel ellipsoidea, glabra, 4n0–4n5i2n5–6n0 µm ; primum alba deinde citrinoviridescentia. Chlamydosporae abundantes.
Typus : Brazil : Para! , Belem, from dead cocoa broom, C. N. Bastos, TVC, GJS 97-183 (BPI–dried culture–holotypus ; live cultures : ATCC 204426, CBS 101875 ; ITS sequence : AF097913). Optimum temperature for growth : 25m. Radius of colonies on PDA after 48 h at 25m 29p1n1 mm. After 72 h mycelium dense, appressed, off-white, with conidia forming in a dense white shelf around the centre and more or less dense broad band around the margin of the colony (dependent on the strain). Conidia white, slowly becoming yellow, green, or yellow-green (3B–E8 or 27C4 and then in the middle of the colony). Radius of colonies on SNA after 64 h at 25m 10n5p2n5 mm. After 72 h aerial mycelium lacking, conidia formed abundantly in 1–3 mm diam. pulvinate to hemispherical pustules. Conidia grey-green (27E4–5). Colonies grown 7 d on CMD at ca 25m in ambient light and for 14 d on oatmeal agar (Difco) and malt extract agar (Difco) at 20m in alternating darkness and cool white fluorescent light 9 cm diam. No aerial mycelium, diffusing pigment or odour. Conidia forming in discrete 1–2 mm diam pustules with little or no conidial production from mononematous conidiophores apart from the pustules. Conidia on all media slowly becoming green (26E5–6) on oatmeal agar and CMD but remaining cream or more yellow after 2 wk on malt extract agar. Pustules compact and very easily dislodged from
G. J. Samuels and others
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Figs 2–13. Trichoderma stromaticum. Figs 2, 3. Conidial pustules formed on CMD. In Fig. 3 terminally fertile extensions of conidiophores are visible. Fig. 4. Anatomy of conidial pustule showing subglobose cells at the interior and conidiophores arising from them. Fig. 5. A conidial pustule with protruding, terminally fertile extensions of conidiophores. Fig. 6. Conidiophore with clustered phialides. Fig. 7. Conidiophore with phialides clustered on short lateral branches at the base of a terminally fertile extension of the conidiophore. Figs 8, 9. Mononematous conidiophores formed apart from the conidial pustules. Fig. 10. Conidia. Figs 11–13. Chlamydospores. All taken from CMD. Figs 2, 3, 6, 12 from GJS 97-180 ; 4, 8, 9 from GJS 97-179 ; 5, 7, 10, 11, 13 from GJS 97-181. Scale bars : Figs 2, 3 l 0n5 mm ; 4, 6, 7, 9, 10 l 10 µm ; 5, 8 l 20 µm ; 11–13 l 25 µm.
Trichoderma stromaticum, sp. nov. the agar surface, formed of vesicular or pseudoparenchymatous, thin-walled, 4n0–4n5 µm wide cells arranged in chains that tend to branch dichotomously near the surface of the pustule. Phialides clustered on terminal cells of the chains of cells, subglobose to ampulliform and sharply constricted toward the tip, 5n5–7n0 µm long, 3n5–4n0 µm in the middle, 3n0–3n5 µm at the base. Awl-like, terminally fertile, ca 50 µm long extensions of conidiophores conspicuously arising from each pustule, each ca 5 µm wide at the base and bearing a single, terminal, ca 15 µm long, subulate phialide tapering from ca 3 µm wide at the base. Often short branches bearing ampulliform phialides arising from near the base of the long conidiophores. Conidia oblong to ellipsoidal, 4n0–4n5i2n5– 3n0 µm, smooth, lacking a visible basal abscission scar. Chlamydospores abundantly formed, globose to subglobose, ca 8 µm diam, terminal and intercalary in hyphae, solitary or aggregated into clusters. Known distribution : Brazil (Rondo! nia, Para! ). Additional cultures examined : Brazil : Para, Belem, from dead cocoa broom, C. N. Bastos, T016 l GJS 97-181 (BPI, ATCC 204427, AF097910) ; Belem, from dead cocoa broom, C. N. Bastos, T0131, GJS 97-182 (BPI, ATCC 204428, AF097912) ; Altamira, from dead cocoa broom, C. N. Bastos, TALT, GJS 97-179 (BPI, ATCC 204429, AF098287). Ouro Preto : Rondo! nia, from soil, A. C. Mendes 1504, GJS 97-180 (BPI, ATCC 204425, AF097911). A C K N O W L E D G E M E N TS G. J. S. and R. P-D. were supported in part by United States National Science Foundation PEET grant to the Pennsylvania State University, ‘ Monographic Studies of Hypocrealean Fungi : Hypocrea and Hypomyces ’ (NSF-97-12308). Dr Walter Gams corrected the Latin description. Ms Tara Osborne and Mr James Plaskowitz provided superb technical assistance.
REFERENCES Bastos, C. N. (1988) Resultados preliminares sobre a efica! cia de Trichoderma viride no controle da vassoura-de-bruxa (Crinipellis perniciosa) do cacaueiro. Fitopatologia Brasileira 13 : 340–342. Bastos, C. N. (1996 a) Mycoparasitic nature of the antagonism between Trichoderma viride and Crinipellis perniciosa. Fitopatologia Brasileira 21 : 50–54. Bastos, C. N. (1996 b) Effect of water potential on growth and viability of conidia of Trichoderma viride after storage at different temperatures. Summa Phytopathologia 22 : 258–261. Bastos, C. N. (1996 c) Potencial de Trichoderma viride no controle da vassourade-bruxa (Crinipellis perniciosa) do cacaueiro. Fitopatologia Brasileira 21 : 509–512.
764 Bissett, J. (1991) A revision of the genus Trichoderma. III. Section Pachybasium. Canadian Journal of Botany 69 : 2373–2417. Costa, J. C. B. & Bezerra, J. L. (1994) Antagonismo de isolados de Cladosporium, Gliocladium, Penicillium, Streptomyces e Trichoderma sobre Crinipellis perniciosa na regia4 o cacueira da Bahia. 4th Siconbiol SimpoT sio de controle bioloT gico de doencm as de Plantas Anais : Sessag o de PoV steres. 59 (abstract). Costa, J. C. B., Bezerra, J. L. & Cazorla, I. M. (1996) Controle biolo! gico da vassoura-de-bruxa do cacueiro na Bahia com Trichoderma polysporum. Fitopatologia Brasileira 21 (Suppl.) : 397 (abstract). Felsenstein, J. (1985) Confidence limits on phylogenies : an approach using the bootstrap. Evolution 39 : 783–791. Kindermann, J., El-Ayouti, Y., Samuels, G. J. & Kubicek, C. P. (1998) Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster. Fungal Genetics and Biology 24 : 298–309. Kornerup, A. & Wanscher, J. H. (1978) Methuen Handbook of Colour. Methuen, London. Kuhls, K., Lieckfeldt, E., Samuels, G. J., Bo$ rner, T., Meyer, W. & Kubicek, C. P. (1997) Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89 : 442–460. Lieckfeldt, E., Samuels, G. J., Bo$ rner, T. & Gams, W. (1998) Trichoderma koningii : neotypification and Hypocrea teleomorph. Canadian Journal of Botany 76 : 1507–1522. Lieckfeldt, E., Samuels, G. J. & Nirenberg, H. I. (1999) What is Trichoderma viride ? – A morphological and molecular perspective. Applied and Environmental Microbiology 65 : 2418–2428. Nirenberg, H. I. (1976) Untersuchungen u$ ber die morphologische und biologische Difrenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologische Bundesanstalt fuW r Land- und Forstwirtschaft Berlin-Dahlem 169 : 1–117. Raper, C. A. & Raper, J. R. (1972) Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64 : 1088–1117. Rehner, S. A. & Samuels, G. J. (1994) Taxonomy and phylogeny of Gliocladium analyzed by large subunit rDNA sequences. Mycological Research 98 : 625–634. Rehner, S. A. & Samuels, G. J. (1995) Molecular systematics of the Hypocreales : a teleomorph gene phylogeny and the status of their anamorphs. Canadian Journal of Botany 73 (Suppl. 1) : S816–S823. Rehner, S. A. & Uecker, F. A. (1994) Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis. Canadian Journal of Botany 72 : 1666–1674. Samuels, G. J., Lieckfeldt, E. & Nirenberg, H. I. (1999) Description of T. asperella sp. nov. and comparison to T. viride. Sydowia 51 : 71–88. Swofford, D. L. (1991) PAUP : phylogenetic analysis using parsimony, 3;1;1. Computer program distributed by Illinois Natural History Survey, Champaign, IL. Webster, J. (1964) Culture studies on Hypocrea and Trichoderma I. Comparison of perfect and imperfect states of H. gelatinosa, H. rufa and Hypocrea sp. 1. Transactions of the British Mycological Society 47 : 75–96. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols : a Guide to Methods and Applications (M. Innis, D. Gelfand, J. Sninsky & T. White, eds) ; 315–322. Academic Press, San Diego. Corresponding Editor : P. M. Kirk
Mycol. Res. 104 (6) : 760–764 (June 2000). Printed in the United Kingdom.
Trichoderma stromaticum sp. nov., a parasite of the cacao witches broom pathogen
G. J. SAMUELS1*, R. PARDO-SCHULTHEISS1, K. P. HEBBAR2, R. D. LUMSDEN2, C. N. BASTOS3, J. C. COSTA4 and J. L. BEZERRA4 " United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Laboratory, Room 304, B-011A, BARC-W, Beltsville, MD 20705, USA. # United States Department of Agriculture, Agricultural Research Service, Biocontrol of Plant Diseases Laboratory, B-011A, BARC-W, Beltsville, MD 20705, USA. $ CEPLAC\SUPOR, Rodovia Augusto Montenegro, Km 7, Belem-Para, CEP 66635-110, Brazil. % CEPLAC\CEPEC – Secag o de Fitopatologia, C. Postal 7, Itabuna, Bahia, CEP 45600-000, Brazil. E-mail : gary!fungi.ars-grin.gov Received 2 February 1999 ; accepted 20 August 1999.
A new species, Trichoderma stromaticum, was isolated from ‘ witches’ broom ’ of cacao (Theobroma cacao) in Brazil caused by Crinipellis perniciosa. The Trichoderma was reported earlier as T. viride and T. polysporum. Trichoderma stromaticum reduced new inoculum through the suppression of basidioma formation. It is characterised by having conidia that slowly become yellow-green in agar culture and by stout, ‘ Pachybasium-like ’, phialides formed on the surface of stromatic structures. The closest relations of T. stromaticum are T. harzianum and T. virens.
INTRODUCTION ‘ Witches’ broom ’ of cacao (Theobroma cacao), caused by the basidiomycete Crinipellis perniciosa, is one of the major problems of cacao culture in tropical South America and the Caribbean islands (Bastos 1988). Symptoms of this disease include the formation of numerous small branches, termed brooms, from the infected meristems and flower cushions. There is no effective means of controlling the disease, although phytosanitation by removal of inoculum that forms from infected tissue in the brooms limits spread of the disease. Chemical controls have not proven effective but within the last ten years Bastos, Costa and their collaborators (Bastos 1988, 1996 a, b, c, Costa & Bezerra 1994, Costa, Bezzera & Cazorla 1996) have isolated a species of Trichoderma from dry brooms of cacao collected in Belem, Brazil. When applied to dry infected brooms, the Trichoderma prevented the formation of new inoculum through the suppression of basidioma formation. Moreover, no mycelium of the pathogen grew out of naturally infected brooms that were inoculated with conidia of the Trichoderma and then incubated in a damp chamber ; it was concluded that the Trichoderma was a direct parasite on mycelium of the pathogen (Bastos 1996 a). This Trichoderma has been misidentified and reported in the literature as T. viride (Bastos 1988, 1996 a, b, c, Costa & Bezerra 1994, and T. polysporum (Costa et al. 1996). Critical examination of the reported strains, however, reveals them to
be of an undescribed species of Trichoderma. Morphologically, it differs from T. viride (see Webster 1964, Lieckfeldt, Samuels & Nirenberg 1999, Samuels, Lieckfeldt & Nirenberg 1999) most conspicuously in forming smooth conidia from discrete, almost stromatic structures. Furthermore, the pattern of branching of conidiophores of this species is very different from what is found in T. viride. There is some similarity to T. polysporum (see Bissett 1991), in that both produce their conidia on discrete, cushion-shaped pustules. The similarity is only superficial, however, since conidia of T. polysporum are white to cream-coloured, only becoming at most pale green or grey with age, whereas conidia of the isolates from cacao are white for a long time but ultimately become green or yellow. Moreover, the pustules of T. polysporum are formed of hyphal elements that are relatively easily teased apart, while the pustules of the cacao isolates are very compact and formed of pseudoparenchymatous cells with conidia arising from conidiophores at the surface. The pustules of the cacao isolates are very compact and are only loosely attached to the agar surface ; they are easily dislodged en masse when the edge of the petri dish is tapped and themselves could possibly act as inoculum. The shape and arrangement of phialides indicate that the cacao isolates belong to Trichoderma sect. Pachybasium (Bissett 1991) but differ considerably from all species that were included in that section. It is, therefore, described below as T. stromaticum. Kindermann et al. (1998) found that Trichoderma
G. J. Samuels and others
761
sect. Pachybasium is paraphyletic with respect to the ITS-1 region of rDNA and that some species that have a typical Pachybasium-like arrangement of phialides are more closely related to T. viride and other more typical species of Trichoderma than they are to each other. Thus, the morphology of T. stromaticum is not sufficient to predict its relationships. In an effort to determine the relationships of this fungus, analysis was undertaken of the ITS rDNA sequences in comparison with other species of Trichoderma.
above. Growth rates on potato dextrose agar (PDA, Difco) and ‘ special nutrient agar ’ (SNA, Nirenberg 1976) were determined following the scheme of Lieckfeldt et al. (1998). Colours are taken from Kornerup & Wanscher (1978). Sequences and fungal strains Sequences used in this study were obtained through GenBank. The isolates from cacao, GJS 97-179–97-183, from Brazil, are listed in Table 1.
MATERIALS AND METHODS Morphological observations
Nucleic acid extraction
All morphological observations are taken from cultures grown on cornmeal dextrose agar (CMD, Difco cornmeal agarj2 % glucose) at 20–21 mC, alternating cool white fluorescent light and dark. Conidial pustules were placed in a drop of 3 % KOH on a microscope slide and gently teased apart with needles. A cover slip was applied and observations were made with a light microscope. Water was added as necessary to prevent mounts from drying. Thirty of each morphological parameter were measured. Measurements are reported as the maximum and minimum of the means of the five collections of T. stromaticum.
Isolates of Trichoderma virens and T. stromaticum GJS 97-179–97-183 were grown on CMD plates at 20m for approximately one week. At this time, a 5 mm diam. agar plug was removed from the edge of each colony and placed in 15 ml liquid CYM (Raper & Raper 1972). Liquid cultures were maintained in darkness at room temperature for approximately 5 d, at which time the mycelial mat was transferred to sterile filter paper, blotted dry and frozen at k80m followed by lyophilization. The lyophilized mycelium was ground with a sterile applicator stick using liquid nitrogen in 1n5 ml microcentrifuge tubes. DNA extraction was performed following Rehner & Uecker (1994) and Rehner & Samuels (1994, 1995).
Cultural observations Morphology of colonies and conidial pustules was described from cultures grown on CMD under the conditions noted Table 1. Strains of Hypocrea and Trichoderma used and their GenBank accession numbers.
H. aureoviridis H. pachybasioides H. pilulifera T. atroviride T. hamatum T. hamatum T. harzianum T. inhamatum T. inhamatum T. koningii T. polysporum T. stromaticum T. stromaticum T. stromaticum T. stromaticum T. stromaticum T. virens T. virens T. viride T. viride
Strain
GenBank no.
CBS" 245.63 ATCC# 18650 CBS 814.68 DAOM$ 165779 ATCC 28012 DAOM 167057 CBS 819.68 CBS 273.78 CBS 518.81 CBS 459.96 CBS 820.68 GJS% 97-179 GJS 97-183 GJS 97-181 GJS 97-182 GJS 97-180 GL& 20 GLi& 39 CBS 240.63 GJS 89-127
Z48819 AF011929 Z48813 Z48817 X93975 Z48816 Z68189 Z68187 Z68188 Z79628 Z48815 AF098287' AF097913' AF097910' AF097912' AF097911' AF099007' AF099005' X93979 X93980
" CBS : Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. # ATCC : American Type Culture Collection, Manassas, VA, USA. $ DAOM : Eastern Cereals and Oilseeds Research Centre, Agriculture and
Agri-Food Canada, Ottawa, Ontario, Canada. % GJS : Collection of G. J. Samuels, Systematic Botany and Mycology Laboratory, US Dept of Agriculture, Beltsville, MD, USA. & GL, GLi : Biocontrol of Plant Diseases Laboratory, US Dept of Agriculture, Beltsville, MD, USA. ' Newly deposited sequences.
PCR and DNA sequencing PCR was used to amplify the ITS region of T. virens and the four cacao isolates. Amplification was performed in a 50 µl reaction volume using 5 µm of template DNA, 10i PCR buffer (Perkin Elmer\ABI, Foster City, CA), 200 m total dNTPs, 3 m MgCl , 1n25 Units Amplitaq Gold4 (Perkin # Elmer\ABI) and 25 pmole each of primers ITS5 and ITS4 (White et al. 1990). The PCR reaction mix was placed in a 0n2 ml microcentrifuge tube, the PCR carried out on a GeneAmp2 9700 PCR System (Perkin Elmer\ABI) and the samples amplified according to the following parameters : 10 min initial activation of Amplitaq Gold2 at 95m, denaturation for 30 s at 93m, annealing for 30 s at 55m, extension for 2 min at 72m for 35 cycles and a 10 min final extension period at 72m. Following amplification, residual nucleotides and primers were removed using the QIAquick4 PCR purification kit (QIAGEN Inc.) according to the manufacturer’s instructions and the PCR purified product eluted in QIAGEN elution buffer (10 m Tris.Cl, pH 8n5). The PCR templates were sequenced using the dRhodamine terminator cycle sequencing kit (Perkin Elmer\ABI) according to the manufacturer’s protocol. Sequenced product purification of unicorporated dye terminators from the extension products were removed with 70 % ethanol and 0n5 m MgCl (1000 : 1, # v\v) according to the manufacturer’s instructions. Extension products were eluted in 15 µl of template suppression reagent (TSR4) (Perkin Elmer\ABI) and analyzed on a model 310 fluorescent automated sequencer (Perkin Elmer\ABI). Sequences were determined on both strands using the following sequencing primers : ITS5, ITS4, ITS2 internal reverse and ITS3 internal forward (White et al. 1990).
Trichoderma stromaticum, sp. nov.
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Sequencing and phylogenetic analysis Sequences were edited, joined and aligned using the EditSeq, SeqMan II, and MegAlign modules in the Lasergene sequence software package (DNAStar, Inc.). Phylogenetic relationships were estimated from the aligned sequences and GenBank sequences using PAUP 3;1;1 (Swofford 1991). Maximum parsimony trees were inferred using the heuristic search (random sequence addition) and branch swapping (tree bisection-reconnection) options. All molecular characters were unordered and given equal weight during the analysis. Support for the phylogenetic groupings was obtained with bootstrap analyses (Felsenstein 1985) using 1000 replications with random addition input orders of sequences during each heuristic search. RESULTS The ITS rDNA data set for this study consisted of 17 taxa and approximately 640 bp per taxon, representing a total of 685 characters. For this data set, 76 characters were phylogenetically informative. Of these 76 characters, 45 were in the ITS1 region and 26 were in the ITS2 region. One informative character was in the beginning of the 5h end of the sequence that incorporates approximately 49 nucleotides of the 18S rDNA gene. Parsimony analysis produced four most parsimonious cladograms (Fig. 1) of 117 steps with a consistency index (CI) of 0n812 and a retention index (RI) of 0n925. T. virens (GL-20)
81
T. virens (GLi-39-type) 96
T. harzianum (CBS 819.68) 94
T. inhamatum (CBS 273.78)
76 72
100
T. inhamatum (CBS 518.81)
T. stromaticum (GJS 97-179) T. stromaticum (GJS 97-183-type)
H. aureoviridis (CBS 245.63) H. pilulifera (CBS 814.68) 95 100
H. pachybasioides (ATCC 18650) T. polysporum (CBS 820.68)
T. hamatum (ATCC 28012) H. rufa (GJS 89-127)
61 81 100 100
T. viride (CBS 240.63) T. koningii (CBS 459.96) T. atroviride (DAOM 165779)
T. hamatum (DAOM 167057 ex NT)
Fig. 1. Cladogram of phylogenetic placement of Trichoderma stomaticum. One of four most parsimonious trees (length l 117, CI l 0n812, RI l 0n925) inferred using heuristic and branch swapping options in Paup 3;1;1. All characters were unordered and given equal weight during analysis. Bootstrap support of 1000 replications is listed above the branches.
Bootstrap supports of 1000 replicates are indicated above the branches. The ITS sequence data for the five strains of T. stromaticum revealed that GJS 97-180 to GJS 97-183 had identical sequences while GJS 97-179 had two deletions (nt TT) at positions 206 and 207 and one substitution (nt C) at position 495. These deletions and substitutions were not phylogenetically informative, and all strains were identified as T. stromaticum. Analysis of the ITS region of this data set revealed that T. stromaticum is a terminal sister group to T. virens, T. harzianum and T. inhamatum. The strict consensus of the ITS region revealed high bootstrap values for most of the nodes in the cladogram. Hypocrea aureoviridis split the biocontrol group from the rest of the taxa examined, although this is only a reflection of the uncertainty of the placement of H. aureoviridis in relation to sect. Pachybasium. Groupings were consistent with data expressed in Kuhls et al. (1997) regarding the paraphyly of sect. Pachybasium. These data, however, only attempt to show the placement of T. stromaticum in relation to other Trichoderma species. From the molecular data, T. stromaticum represents a sister taxon to T. harzianum and T. virens. Trichoderma stromaticum Samuels & Pardo-Schultheiss, sp. nov. (Figs 2–13) Lente crescens. Conidia e pustulis 1–3 mm diam, pulvinatis vel subglobularibus exorientia. Cellulae pustularum vesiculares vel pseudoparenchymaticae, tenuitunicatae. Phialides dense dispositae, ex ramulis brevibus vel basim versus ex extensionibus subulatis conidiophororum oriundae, subglobosae vel ampulliformes, 5n5– 7n0 µm longae, 3n5–4n0 µm latae in medio. Conidia oblonga vel ellipsoidea, glabra, 4n0–4n5i2n5–6n0 µm ; primum alba deinde citrinoviridescentia. Chlamydosporae abundantes.
Typus : Brazil : Para! , Belem, from dead cocoa broom, C. N. Bastos, TVC, GJS 97-183 (BPI–dried culture–holotypus ; live cultures : ATCC 204426, CBS 101875 ; ITS sequence : AF097913). Optimum temperature for growth : 25m. Radius of colonies on PDA after 48 h at 25m 29p1n1 mm. After 72 h mycelium dense, appressed, off-white, with conidia forming in a dense white shelf around the centre and more or less dense broad band around the margin of the colony (dependent on the strain). Conidia white, slowly becoming yellow, green, or yellow-green (3B–E8 or 27C4 and then in the middle of the colony). Radius of colonies on SNA after 64 h at 25m 10n5p2n5 mm. After 72 h aerial mycelium lacking, conidia formed abundantly in 1–3 mm diam. pulvinate to hemispherical pustules. Conidia grey-green (27E4–5). Colonies grown 7 d on CMD at ca 25m in ambient light and for 14 d on oatmeal agar (Difco) and malt extract agar (Difco) at 20m in alternating darkness and cool white fluorescent light 9 cm diam. No aerial mycelium, diffusing pigment or odour. Conidia forming in discrete 1–2 mm diam pustules with little or no conidial production from mononematous conidiophores apart from the pustules. Conidia on all media slowly becoming green (26E5–6) on oatmeal agar and CMD but remaining cream or more yellow after 2 wk on malt extract agar. Pustules compact and very easily dislodged from
G. J. Samuels and others
763
Figs 2–13. Trichoderma stromaticum. Figs 2, 3. Conidial pustules formed on CMD. In Fig. 3 terminally fertile extensions of conidiophores are visible. Fig. 4. Anatomy of conidial pustule showing subglobose cells at the interior and conidiophores arising from them. Fig. 5. A conidial pustule with protruding, terminally fertile extensions of conidiophores. Fig. 6. Conidiophore with clustered phialides. Fig. 7. Conidiophore with phialides clustered on short lateral branches at the base of a terminally fertile extension of the conidiophore. Figs 8, 9. Mononematous conidiophores formed apart from the conidial pustules. Fig. 10. Conidia. Figs 11–13. Chlamydospores. All taken from CMD. Figs 2, 3, 6, 12 from GJS 97-180 ; 4, 8, 9 from GJS 97-179 ; 5, 7, 10, 11, 13 from GJS 97-181. Scale bars : Figs 2, 3 l 0n5 mm ; 4, 6, 7, 9, 10 l 10 µm ; 5, 8 l 20 µm ; 11–13 l 25 µm.
Trichoderma stromaticum, sp. nov. the agar surface, formed of vesicular or pseudoparenchymatous, thin-walled, 4n0–4n5 µm wide cells arranged in chains that tend to branch dichotomously near the surface of the pustule. Phialides clustered on terminal cells of the chains of cells, subglobose to ampulliform and sharply constricted toward the tip, 5n5–7n0 µm long, 3n5–4n0 µm in the middle, 3n0–3n5 µm at the base. Awl-like, terminally fertile, ca 50 µm long extensions of conidiophores conspicuously arising from each pustule, each ca 5 µm wide at the base and bearing a single, terminal, ca 15 µm long, subulate phialide tapering from ca 3 µm wide at the base. Often short branches bearing ampulliform phialides arising from near the base of the long conidiophores. Conidia oblong to ellipsoidal, 4n0–4n5i2n5– 3n0 µm, smooth, lacking a visible basal abscission scar. Chlamydospores abundantly formed, globose to subglobose, ca 8 µm diam, terminal and intercalary in hyphae, solitary or aggregated into clusters. Known distribution : Brazil (Rondo! nia, Para! ). Additional cultures examined : Brazil : Para, Belem, from dead cocoa broom, C. N. Bastos, T016 l GJS 97-181 (BPI, ATCC 204427, AF097910) ; Belem, from dead cocoa broom, C. N. Bastos, T0131, GJS 97-182 (BPI, ATCC 204428, AF097912) ; Altamira, from dead cocoa broom, C. N. Bastos, TALT, GJS 97-179 (BPI, ATCC 204429, AF098287). Ouro Preto : Rondo! nia, from soil, A. C. Mendes 1504, GJS 97-180 (BPI, ATCC 204425, AF097911). A C K N O W L E D G E M E N TS G. J. S. and R. P-D. were supported in part by United States National Science Foundation PEET grant to the Pennsylvania State University, ‘ Monographic Studies of Hypocrealean Fungi : Hypocrea and Hypomyces ’ (NSF-97-12308). Dr Walter Gams corrected the Latin description. Ms Tara Osborne and Mr James Plaskowitz provided superb technical assistance.
REFERENCES Bastos, C. N. (1988) Resultados preliminares sobre a efica! cia de Trichoderma viride no controle da vassoura-de-bruxa (Crinipellis perniciosa) do cacaueiro. Fitopatologia Brasileira 13 : 340–342. Bastos, C. N. (1996 a) Mycoparasitic nature of the antagonism between Trichoderma viride and Crinipellis perniciosa. Fitopatologia Brasileira 21 : 50–54. Bastos, C. N. (1996 b) Effect of water potential on growth and viability of conidia of Trichoderma viride after storage at different temperatures. Summa Phytopathologia 22 : 258–261. Bastos, C. N. (1996 c) Potencial de Trichoderma viride no controle da vassourade-bruxa (Crinipellis perniciosa) do cacaueiro. Fitopatologia Brasileira 21 : 509–512.
764 Bissett, J. (1991) A revision of the genus Trichoderma. III. Section Pachybasium. Canadian Journal of Botany 69 : 2373–2417. Costa, J. C. B. & Bezerra, J. L. (1994) Antagonismo de isolados de Cladosporium, Gliocladium, Penicillium, Streptomyces e Trichoderma sobre Crinipellis perniciosa na regia4 o cacueira da Bahia. 4th Siconbiol SimpoT sio de controle bioloT gico de doencm as de Plantas Anais : Sessag o de PoV steres. 59 (abstract). Costa, J. C. B., Bezerra, J. L. & Cazorla, I. M. (1996) Controle biolo! gico da vassoura-de-bruxa do cacueiro na Bahia com Trichoderma polysporum. Fitopatologia Brasileira 21 (Suppl.) : 397 (abstract). Felsenstein, J. (1985) Confidence limits on phylogenies : an approach using the bootstrap. Evolution 39 : 783–791. Kindermann, J., El-Ayouti, Y., Samuels, G. J. & Kubicek, C. P. (1998) Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster. Fungal Genetics and Biology 24 : 298–309. Kornerup, A. & Wanscher, J. H. (1978) Methuen Handbook of Colour. Methuen, London. Kuhls, K., Lieckfeldt, E., Samuels, G. J., Bo$ rner, T., Meyer, W. & Kubicek, C. P. (1997) Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89 : 442–460. Lieckfeldt, E., Samuels, G. J., Bo$ rner, T. & Gams, W. (1998) Trichoderma koningii : neotypification and Hypocrea teleomorph. Canadian Journal of Botany 76 : 1507–1522. Lieckfeldt, E., Samuels, G. J. & Nirenberg, H. I. (1999) What is Trichoderma viride ? – A morphological and molecular perspective. Applied and Environmental Microbiology 65 : 2418–2428. Nirenberg, H. I. (1976) Untersuchungen u$ ber die morphologische und biologische Difrenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologische Bundesanstalt fuW r Land- und Forstwirtschaft Berlin-Dahlem 169 : 1–117. Raper, C. A. & Raper, J. R. (1972) Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64 : 1088–1117. Rehner, S. A. & Samuels, G. J. (1994) Taxonomy and phylogeny of Gliocladium analyzed by large subunit rDNA sequences. Mycological Research 98 : 625–634. Rehner, S. A. & Samuels, G. J. (1995) Molecular systematics of the Hypocreales : a teleomorph gene phylogeny and the status of their anamorphs. Canadian Journal of Botany 73 (Suppl. 1) : S816–S823. Rehner, S. A. & Uecker, F. A. (1994) Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis. Canadian Journal of Botany 72 : 1666–1674. Samuels, G. J., Lieckfeldt, E. & Nirenberg, H. I. (1999) Description of T. asperella sp. nov. and comparison to T. viride. Sydowia 51 : 71–88. Swofford, D. L. (1991) PAUP : phylogenetic analysis using parsimony, 3;1;1. Computer program distributed by Illinois Natural History Survey, Champaign, IL. Webster, J. (1964) Culture studies on Hypocrea and Trichoderma I. Comparison of perfect and imperfect states of H. gelatinosa, H. rufa and Hypocrea sp. 1. Transactions of the British Mycological Society 47 : 75–96. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols : a Guide to Methods and Applications (M. Innis, D. Gelfand, J. Sninsky & T. White, eds) ; 315–322. Academic Press, San Diego. Corresponding Editor : P. M. Kirk