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Crinipellis minima S. A. Sharafudheen, Manim. & K. P. D. Latha, sp. nov. Fig. 2A–L MycoBank no.: MB 847917 Etymology:— minima (L.), small; refers to the very small basidiocarps of this species. Diagnosis:—Differs from C. kisanganensis in having a smaller (up to 20 mm long) stipe covered with orange white, recurved squamules, a hymenium with pleurocystidia, and the lamellar trama with a dextrinoid central region. Type:— INDIA. Kerala State: Thiruvananthapuram District, Neyyar Wildlife Sanctuary, 8°33’24.7” N 77°09’33.0” E, 22 October 2017, A. S. Shahina SA324 (holotype CALI!). GenBank accessions: nrITS: OQ617341 and nrLSU: OQ617343. Description:— Basidiocarps small. Pileus 3–4.5 mm diam., initially convex, becoming convex with a shallow central depression; surface initially dark brown (7F5/OAC700) all over with a darker center (7F8/OAC733), becoming dark brown (7F7/OAC720) at the center, light brown (7D5/OAC701) around it, grayish orange (5B3 to 5B5/OAC813 to OAC814) towards the margin, with brown (7E8/OAC700 or OAC701), radially appressed fibrils extending to the margin; margin straight, appendiculate. Lamellae free or at times adnexed, up to 1 mm wide, light orange white (5A2/OAC900), subclose, with lamellulae of 1 tier; edge torn under a lens, concolorous with the sides. Stipe 11−20 × 0.5–0.75 mm, central, terete, equal; surface initially pale yellow (4A3/OAC805) at the base and the apex and yellowish white (4A2/OAC900) in the middle, becoming light brown (6D4/OAC700) all over except at the apex where it is concolorous to the lamellae when mature, with orange white (5A2/OAC815), recurved squamules all over, densely so towards the base; base insititious. Rhizomorphs not observed. Context thin. Basidiospores 9–10.5 × 4–5 (9.63 ± 0.51 × 4.4 ± 0.48) µm, Q = 1.9–2.5, Qm = 2.20, fusoid to ovo-ellipsoid, smooth, thin-walled, rarely slightly thick-walled, hyaline, inamyloid. Basidia 21–28 × 6–8 µm, clavate, 4-spored; sterigmata up to 3 µm long. Pleurocystidia scattered to rare, not observed in some lamellae, 19–31 × 5–9 µm, fusoid or clavate with a mucronate apex, at times with constrictions in the body, hyaline, thin- to slightly thick-walled. Lamella-edge heteromorphous with abundant cheilocystidia. Cheilocystidia 16–26 × 5–9 µm, resembling Siccus - type broom cells: broadly clavate to subcylindrical, hyaline, thin- to slightly thick-walled; apical appendages up to 8 µm long, often branched, subacute to obtuse, hyaline, thin- to slightly thick-walled, sometimes appendages reduced or with lobes. Lamellar trama subregular; hyphae 4–14 µm wide, thin- to slightly thick-walled (up to 1 µm thick), hyaline, central part of the trama weakly dextrinoid. Pileus trama subregular; hyphae 2–15 µm wide, thin-walled, hyaline, inamyloid. Pileipellis a hypotrichium composed of closely septate, inflated hyphae giving rise to suberect terminal hairs; hypotrichial hyphae 11–16 µm wide, thin- to slightly thick-walled, hyaline or pale yellow, hyphae brown at the central part of the trama in water and 5% KOH, sometimes with hyaline encrustations; hairs 46–438 × 4–7 µm, cylindrical or flexuous with subacute to obtuse apices, rarely with septa, often with a swollen base, light brown to brown or somewhat grayish in water and 5% KOH, dextrinoid, thick-walled (up to 1.5 µm thick). Stipitipellis similar to the pileipellis but with narrow hypotrichial hyphae and short hairs; hypotrichial hyphae 2.5–7 µm wide, thick-walled (up to 1 µm thick), yellowish brown to brown in water, turning greenish in 5% KOH. Stipitipellis hairs 55.5–252 × 5–11 µm, tapering towards the apex, hyaline or yellowish brown, septa not observed. Stipe trama dextrinoid. Clamp connections observed on all hyphae. Habitat: — Scattered, on decaying dicotyledonous leaves and twigs. Geographical distribution range:—Known only from the type locality in Kerala State, India. Comments:—Small basidiocarps, a pileus with a central depression, a stipe without rhizomorphs, a hymenium with cheilo- and pleurocystidia, a weakly dextrinoid middle region of lamellar trama, pileipellis hyphae (hypotrichium) lacking discoloration in KOH and stipitipellis hyphae (hypotrichium) turning greenish in KOH are the characteristic features of this species. Pleurocystidia were found to be scattered or rare or absent in different specimens of the same collection. Crinipellis kisanganensis Antonín & Buyck (in Antonín 2012: 402), a species described from Congo and Zimbabwe (Antonín 2012), is comparable to C. minima in having small basidiocarps, a pileus with a dark brown depressed center, basidiospores of almost similar size (8–10 × 3.5–4.75 µm), cheilocystidia of almost similar size ((12) 15–26 × 4.5–9 µm) and morphology and the pileipellis hairs not changing color in KOH. However, C. kisanganensis is distinct from C. minima in having a longer (up to 35 mm long) and laterally compressed stipe covered with dark brown hairs or grayish brown hairs, the absence of pleurocystidia and a dextrinoid middle region in the lamellar trama. There is no mention of the reaction of stipitipellis (hypotrichium) with KOH in that collection. Crinipellis dipterocarpi Singer (1942: 496), a species originally described from Vietnam and later from Indonesia, Malaysia, Thailand (Kerekes & Desjardin 2009) and Japan (C. dipterocarpi f. cinnamomea Kerekes, Desjardin & Lumyong, (in Kerekes & Desjardin 2009: 120)), is also similar to C. minima in having small basidiocarps, a convex pileus with a brown center, cheilocystidia with apical appendages, the pileipellis hairs not turning green in KOH and the stipitipellis hyphae turning green in KOH. However, C. dipterocarpi has adnate to adnexed lamellae, a longer stipe (up to 50 mm long), the occasional presence of rhizomorphs, inamyloid lamellar trama, a hymenium devoid of pleurocystidia and the pileipellis hyphae with reddish brown encrustations (Kerekes & Desjardin 2009). A pairwise comparison of the nrITS sequences of various GenBank accessions of C. dipterocarpi (FJ167656, FJ167651, FJ167655, FJ167648, FJ167653, FJ167652, FJ167650, FJ167654, FJ167649) with that of C. minima showed only less than 91% sequence similarity. Crinipellis minima is also similar to C. pseudostipitaria var. pseudostipitaria Singer (1942: 470), documented from the neotropics (Singer 1976), in having a pileus with a depressed center, subclose lamellae, basidiospores of almost similar size and shape, presence of cheilocystidia with apical appendages and the pileipellis hairs with scattered septa. However, C. pseudostipitaria var. pseudostipitaria differs from C. minima in having a larger (up to 13 mm broad), fibrillose-woolly pileus in almost uniform color, longer and broader stipe (9–30 × 0.3–2 mm), a hymenium devoid of pleurocystidia and inamyloid lamellar trama. A BLASTn search using the nrITS sequence (626 bp) of C. minima showed Crinipellis nigricaulis var. macrospora Antonín, Ryoo & H.D. Shin (2009: 431) (FJ573197) as the closest taxon with 89.92% similarity. While using nrLSU (896 bp) sequence, C. nigricaulis Har. Takah. (2000: 178) (MK277894: 99.44%) resulted as the closest hit. Similar to C. minima, C. nigricaulis has narrow lamellae up to 1 mm broad, the presence of cheilocystidia with apical appendages and a dextrinoid stipe trama. But C. nigricaulis can be distinguished from C. minima in having a longer stipe (50 mm long) with rhizomorphs, the absence of pleurocystidia, and longer pileipellis hairs (up to 1500 µm long) with numerous secondary septa forming ladder-structure (Takahashi 2000). Crinipellis nigricaulis var. macrospora also has a longer (up to 200 mm long) stipe and a hymenium devoid of pleurocystidia (Antonín et al. 2014)., Published as part of Sharafudheen, Shahina A., Manimohan, Patinjareveettil & Deepna Latha, K. P., 2023, Two new species of Crinipellis (Marasmiaceae, Agaricales) from Kerala State, India, pp. 219-229 in Phytotaxa 600 (4) on pages 223-225, DOI: 10.11646/phytotaxa.600.4.1, http://zenodo.org/record/8093955, {"references":["Antonin, V. (2012) Chaetocalathus and Crinipellis (Basidiomycota, Marasmiaceae) in tropical Africa: taxonomic and nomenclatural novelties. Cryptogamie Mycologie 33 (4): 395 - 410. https: // doi. org / 10.7872 / crym. v 33. iss 4.2012.395","Singer, R. (1942) A Monographic Study of the Genera Crinipellis and Chaetocalathus. Lilloa 8: 441 - 534.","Kerekes, J. F. & Desjardin, D. E. (2009) A monograph of the genera Crinipellis and Moniliophthora from Southeast Asia including a molecular phylogeny of the nrITS region. Fungal Diversity 37: 101 - 152.","Singer, R. (1976) Marasmieae (Basidiomycetes-Tricholomataceae) monograph no. 17, Flora Neotropica. The New York Botanical Garden, New York, 348 pp.","Takahashi, H. (2000) Three new species of Crinipellis found in Iriomote Island, southwestern Japan, and central Honshu, Japan. Mycoscience 41 (2): 171 - 182. https: // doi. org / 10.1007 / bf 02464328","Antonin, V., Ryoo, R., Ka, K. H. & Sou, H. D. (2014) Three new species of Crinipellis and one new variety of Moniliophthora (Basidiomycota, Marasmiaceae) described from the Republic of Korea. Phytotaxa 170 (2): 86 - 102. https: // doi. org / 10.11646 / phytotaxa. 170.2.2"]}

Crinipellis fibrillosa S. A. Sharafudheen, Manim. & K. P. D. Latha, sp. nov. Fig. 3A–N MycoBank no.: MB 847918 Etymology:— fibrillosa (L.), with fibrils; refers to the fibrillose pileus surface of this species. Diagnosis:—Differs from C. tucumanensis in having a pileus with radial rows formed by appressed fibrils, a longer stipe (up to 64 mm long), longer and narrow basidiospores (11–12 (–13) × 3–4 µm) and shorter cheilocystidia (up to 27 µm long). Type:— INDIA. Kerala State: Thrissur District, Kalasamala sacred grove, 10°40’18.7” N 76°05’18.8” E, 10 June 2017, A. S. Shahina SA199 (holotype CALI!). GenBank accessions: nrITS: OQ617333 and nrLSU: OQ617342. Description:— Basidiocarps small. Pileus 5–10 mm diam., convex when young, becoming plano-convex to almost applanate with an occasional papilla visible in dried specimens under a lens; surface dark brown (7F8/OAC635) at the center, brown (7E8/OAC621) around it, reddish gray (7B2/OAC620) to grayish red (7B3/OAC634) towards the margin, with dark brown (7F8/OAC622) radially appressed fibrils, densely so at the center, often forming tufts in radial rows towards the margin and hanging from the margin; margin straight, appendiculate. Lamellae free, up to 2 mm wide, orange-white (5A2/OAC900) close; edge entire to the naked eye, finely torn under a lens, initially concolorous with the sides but in mature specimens the edge becomes dark brown (7F4/OAC737) on drying starting from the part close to the stipe and gradually spreading to the entire edge. Stipe 28–64 × 1 mm, central, terete, equal, solid; surface dark brown (6F8/OAC635), appressed-fibrillose all over; base insititious. Rhizomorphs not observed. Context very thin. Basidiospores 11–12 (–13) × 3–4 (11.83 ± 0.47 × 3.43 ± 0.37) µm, Q = 2.75–4.0, Qm = 3.49, lanceolate, smooth, thin-walled, hyaline, inamyloid. Basidia 22.5–27 × 7–8.5 µm, clavate, 4-spored; sterigmata up to 5.5 µm long. Pleurocystidia 34–43 × 7.5–10 µm, scattered, clavate, elongate-clavate or subcylindrical, hyaline, thin- to slightly thick-walled. Lamella-edge heteromorphous. Cheilocystidia 17–27 × 4–9 µm, versiform: broadly fusiform, subcylindrical, obclavate, vesiculose or flexuous, often with a mucronate apex or bifurcating apical appendages up to 12 µm long, hyaline in young specimens, but with dark brown plasmatic pigment in mature specimens, turning greenish gray in 3% KOH, thin- to slightly thick-walled. Lamellar trama subregular; hyphae 3–17 µm wide, thin- to slightly thick-walled, hyaline, inamyloid. Pileus trama a duplex; upper half with narrow, parallelly interwoven 3–17 µm wide hyphae; lower half with inflated 32–74 × 11–22.5 µm wide, closely septate hyphae, thin- to slightly thick-walled, hyaline to pale yellow, inamyloid. Pileipellis a hypotrichium composed of closely septate, inflated hyphae giving rise to suberect terminal hairs; hypotrichial hyphae 6–13 µm wide, thin- to slightly thick-walled, hyaline or with a brownish wall pigment, turning grayish to grayish green in 3% KOH; hairs 80–555 × 4–8 µm, cylindrical or flexuous, with subacute to obtuse apices, often with septa, brown to light brown or hyaline, turning grayish to grayish green in 3% KOH, dextrinoid, thick-walled (up to 2.5 µm thick). Stipitipellis similar to the pileipellis but with narrow hypotrichial hyphae measuring 2–6 µm wide, hyaline or yellowish brown in water and 3% KOH, thick-walled (up to 1 µm thick); hairs 75–415 × 6–10.5 µm, narrowly cylindrical or at times tapering towards the apex, often branched, septate, hyaline or yellowish brown, turning grayish to grayish green in 3% KOH, thick-walled (up to 2 µm thick). Stipe trama dextrinoid. Clamp connections observed on all hyphae except at the base of basidia, cheilo- and pleurocystidia. Habitat: — Scattered on leaf litter, partially buried in the soil. Geographical distribution range:—Known only from the type locality in Kerala State, India. Comments:— Crinipellis fibrillosa has a pileus with dark brown appressed fibrils, free lamellae, a lamella-edge with cheilocystidia, a hypotrichium-type pileipellis giving rise to long, thick-walled, dextrinoid hairs turning grayish to grayish green in 3% KOH and a habitat on partially buried leaf litter. An exhaustive literature survey showed that the lamella-edge of no other described species of Crinipellis turns dark brown on drying or has the greenish gray coloration of dried lamella-edge in KOH. Crinipellis tucumanensis Singer (1976: 40), a species originally described from Argentina (Singer 1976), shows some similarities to C. fibrillosa in having a pileus of similar size (4–13 mm diam.), free lamellae, pleurocystidia of similar size and shape, a lamella-edge with cheilocystidia and the pileipellis hairs turning greenish in KOH. However, C. tucumanensis can be readily distinguished from C. fibrillosa as the former has a pileus lacking radial rows formed by appressed fibrils, a shorter stipe (up to 36 mm long), shorter and broader basidiospores (5.5–8.5 × 4–6 µm) and longer cheilocystidia (up to 40 µm long). Crinipellis brunneoaurantiaca Bandala, Montoya & Ryoo (in Bandala et al. 2012: 734), described from Mexico, is another species that shows the characteristic greenish color change of pileus hairs in KOH (Bandala et. al. 2012). That species shares some features such as pileus of similar size (2–9 mm diam.) and shape with an appendiculate margin, free lamellae, presence of cheilocystidia, septate pileipellis hairs and a dextrinoid stipe trama with C. fibrillosa. However, C. brunneoaurantiaca has a pileus with a conical to subacute central papilla, subdistant to distant lamellae, a shorter stipe (4–14 mm long) and a hymenium devoid of pleurocystidia. Crinipellis fibrillosa shows some features similar to C. calderi Pegler (1966: 106), a species described from Uganda (Pegler 1977), in having an initially convex and finally expanded-plane pileus with hairs sparser towards the margin, basidiospores of somewhat similar size (9–11.7 × 3.2–4.5 µm), a heteromorphous lamella-edge with cheilocystidia and septate pileipellis hairs. However, C. calderi is distinct from C. fibrillosa in having a radially sulcate pileus, distant and intervenose lamellae, a shorter stipe (up to 35 mm long), lamellae devoid of pleurocystidia and hypotrichial hyphae with incrusting pigments. Crinipellis fibrillosa is also similar to C. malesiana Kerekes, Desjardin & Vikinesw. (in Kerekes & Desjardin 2009: 125), a species from Southeast Asia, having a pileus of similar size (2–13 mm diam.) and shape, a hymenium with pleuro- and cheilocystidia, and a pileipellis with hyaline or brown hyphae turning greenish in KOH. However, this species differs from C. fibrillosa in having a pileus often with one or two raised concentric ridges surrounding a central papilla and a brown to brownish orange margin, adnexed to adnate lamellae, a shorter stipe (4–22 mm long), broader (4–6.5 µm) basidiospores, longer ((11–) 21–40 µm) cheilocystidia and a stipitipellis with caulocystidia (Kerekes & Desjardin 2009). A pairwise comparison of the nrITS sequence (NR_ 119706) of C. malesiana with that of C. fibrillosa showed only 83.59% similarity A BLASTn search of the GenBank nucleotide database using the nrITS sequence (661 bp) of C. fibrillosa showed an unnamed Crinipellis species, Crinipellis species RAK 391 (MN930626) as the closest hit with 88.75% sequence similarity. Crinipellis pseudosplachnoides (Hennings 1901: 47) Pat. ex Singer (1942: 510) (MK277895: 98.78%) resulted as the closest hit using nrLSU (903 bp) sequence. Crinipellis pseudosplachnoides is similar to C. fibrillosa in having a pileus with a brown center, an insititious stipe, a hymenium with cheilo- and pleurocystidia and the pileipellis hairs with septations. But C. pseudosplachnoides has a shorter stipe (up to 40 mm long), shorter and broader (6.3–10 × 3.5–5.7 µm) basidiospores and longer cheilo- (up to 45 µm long) and pleurocystidia (65 µm long) (Pegler 1977)., Published as part of Sharafudheen, Shahina A., Manimohan, Patinjareveettil & Deepna Latha, K. P., 2023, Two new species of Crinipellis (Marasmiaceae, Agaricales) from Kerala State, India, pp. 219-229 in Phytotaxa 600 (4) on pages 225-227, DOI: 10.11646/phytotaxa.600.4.1, http://zenodo.org/record/8093955, {"references":["Singer, R. (1976) Marasmieae (Basidiomycetes-Tricholomataceae) monograph no. 17, Flora Neotropica. The New York Botanical Garden, New York, 348 pp.","Bandala, V. M., Ryoo, R., Montoya, L. & Ka, K. - H. (2012) New species and new records of Crinipellis from tropical and subtropical forests of the east coast of Mexico. Mycologia 104 (3): 733 - 745. https: // doi. org / 10.3852 / 11 - 223","Pegler, D. N. (1966) Tropical African Agaricales. Persoonia 4 (2): 73 - 124.","Pegler, D. N. (1977) A preliminary Agaric Flora of East Africa. Kew Bulletin Additional Series 6: 1 - 615.","Kerekes, J. F. & Desjardin, D. E. (2009) A monograph of the genera Crinipellis and Moniliophthora from Southeast Asia including a molecular phylogeny of the nrITS region. Fungal Diversity 37: 101 - 152.","Hennings, P. (1901) Fungi camerunenses novi. III. Botanische Jahrbucher fur Systematik, Pflanzengeschichte und Pflanzengeographie 30: 39 - 57.","Singer, R. (1942) A Monographic Study of the Genera Crinipellis and Chaetocalathus. Lilloa 8: 441 - 534."]}

The goal of the present research was to ascertain the potential root endophytic fungi against Dematophora necatrix, Hartig, the causal pathogen of white root rot in apples; however, it has an expanded range of hosts across different fruit trees. Out of 36 endophytic fungi segregated from symptomless roots of apple, wheat, maize, marigold, cherry, and garlic plants, only 9 isolates showed inhibitory effect during preliminary screening and were further assayed under in vitro, pot, and field conditions against the white root rot pathogen/disease. Under in vitro conditions, maximum mycelial inhibition of 81.48% was obtained with the isolate Aspergillus aculeatus strain C2. Microscopic studies on interaction between fungal endophyte with hyphal tips of D. necatrix revealed various morphological abnormalities in the hyphae. In glasshouse conditions, seed treatment pursued by soil application with Crinipellis tabtim strain M8 isolate was highly effective and exhibited 93.55% disease control. Similarly, under field conditions, the overall maximum disease control was exhibited by Crinipellis tabtim strain M8 (84.95%). The most promising root endophytes that were identified rely on morphological and ITS sequence analysis. Root colonization assay was performed which revealed maximum endosphere and rhizosphere colonization with Crinipellis tabtim strain M8. Additionally, confocal microscopic illustrations of transverse sections of root cells tenanted by fungal endophytes as compared to untreated control suggested the persistence and establishment of endophytes in the endosphere of apple seedlings. These findings present the first report on colonization of apple roots by fungal root endophytes suggesting an alternative and sustainable approach for management of white root rot disease.

In tropical and subtropical rainforests, vegetative fungal rhizomorphs from the Marasmiineae are routinely used as construction material in bird nests. Because rhizomorphs seldom produce mushrooms within nests, the fungal species involved remain largely unknown. In turn, this limitation has prevented us from resolving broader questions such as whether specific fungal species are selected by birds for different functional roles (i.e., attachment, or parasite control). To fill some of these gaps, we collected 74 rhizomorph-containing bird nests from the Neo- and Afrotropics and used nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) sequences to discriminate between rhizomorph-forming species. In total we recovered 25 Marasmiineae species used by birds in nest construction, none of which were shared between the Neotropics and the Afrotropics. We also collected Marasmiineae basidiomes in the vicinity of nests and used ITS sequences to match these sporulating morphs with nest rhizomorphs for nine species. Basidiomes from an additional five species were found fruiting from rhizomorphs incorporated within bird nests. Finally, an additional six species were putatively identified based on publicly available sequence data. Rhizomorphs of five species were found to be utilized almost exclusively as lining material in nests. Lining material comes in direct contact with nestlings and is hypothesized to play a role in parasite control. Rhizomorphs from 10 species were used to attach and anchor nests to substrates; we matched six of those to fruiting litter trap-forming species collected in the understory. Litter traps hold large quantities of fallen litter material, suggesting that birds may preferentially use rhizomorphs that are adapted to bearing heavy loads for nest attachment. Finally, we describe two species of

The crinipelloid genera Crinipellis and Moniliophthora (Agaricales, Marasmiaceae) are characterized by basidiomes that produce long, dextrinoid, hair-like elements on the pileus surface. Historically most species are believed to be saprotrophic or, rarely, parasitic on plant hosts. The primary morphological diagnostic characters that separate Crinipellis and Moniliophthora are pliant vs. stiff (Crinipellis) stipes and a tendency toward production of reddish pigments (ranging from violet to orange) in the basidiome in Moniliophthora. Additionally, most species of Moniliophthora appear to have a biotrophic habit, while those of Crinipellis are predominantly saprotrophic. Recently, several new neotropical collections prompted a morphological and phylogenetic analysis of this group. Herein, we propose a new species and two new combinations: Moniliophthora mayarum sp. nov., described from Belize, is characterized by its larger pileus and narrower basidiospores relative to other related species; Moniliophthora ticoi comb. nov. (= Crinipellis ticoi) is recollected and redescribed from biotrophic collections from northern Argentina; and M. brasiliensis comb. nov. (= Crinipellis brasiliensis), a parasite of Heteropterys acutifolia. The addition of these three parasitic species into Moniliophthora support a hypothesis of a primarily biotrophic/parasitic habit within this genus. Fil: Niveiro, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Ramírez, Natalia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Michlig, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Lodge, D. Jean. University of Georgia; Estados Unidos Fil: Aime, M. Catherine. Purdue University; Estados Unidos

The trend to search novel natural antifungal compounds has recently been increasing. Interspecific interactions between 30 Macromycetes species and fungal pathogens (Issatchenkia orientalis, Candida albicans strains) have been evaluated using dual culture plate assay. Interaction reactions between studied fungi were different: deadlock after mycelia contact or at a distance, overgrowth without initial deadlock, partial or complete replacement after initial deadlock with contact. Domination of replacement (78.7 %) of pathogenic fungi by Macromycetes was established. Complete replacement was almost twice more frequent (30.7 %) than partial replacement (19.3 %). These results clearly indicate effectiveness of Macromycetes against pathogenic fungi via contact antagonism. Strong antagonistic activity with high antagonistic index (AI) was established by xylotrophic species Ganoderma applanatum (AI = 22.5), Lentinula edodes (AI = 21.0), Flammulina velutipes, Irpiciporus litschaueri and Pleurotus ostreatus (AI = 20.5), leaf litter decay fungus Crinipellis schevczenkoi (AI = 21), and soil saprotroph Lyophyllum shimeji (AI = 21).

The aqueous extracts of Ganoderma lucidum 1900 (Curtis) P. Karst and Crinipellis schevczenkovi 31 Buchalo mycelia were investigated for skin wound healing activity in vivo using the excision wound healing model. The extracts were prepared by mixing of 100 mg of powdered mycelium of both mushrooms with 1 mL of sterile distilled water for injections. White albino mice line FVB/Cg., 3 months of age (male), were used for the study. The rate of wound healing and the histology of healed wounds in mice have been studied. Visual method of wound study and histological investigation of skin tissue showed the presence of wound healing potential of G. lucidum and C. schevczenkovi mycelia. The wound healing process was expressed more active on 3th day in case of topical application of C. schevczenkovi mycelium extract, but on 5th day the wound healing effects of both mushroom extracts were almost at the same level and statistically better then results in the control group. Wounds treated with mycelial extracts were covered completely on the 6th day compared with 8th day in control group. Wound healing ability of C. schevczenkovi observed in present investigations for the first time.

A new species Crinipellis floccosa , discovered from Jiangxi and Guagndong provinces in southern China, is formally introduced. Morphologically the new taxon is similar to C . zonata and C. corvina , but the pale gray to reddish brown floccose squamules on the pileus make it a distinct species, which is also supported by the molecular data based on ribosomal RNA gene ITS sequence. The holotype is deposited in the fungal herbarium of Guangdong Institute of Microbiology (GDGM) and the sequence data is deposited in GenBank.

A number of newly described species, new combinations and new records of New Zealand agaric fungi are introduced together with diagnostic descriptions, phylogenetic position and additional notes. The new species are Clitopilus kamaka, Lyophyllum moncalvoanum, Gerhardtia pseudosaponacea, Clitocybe brunneocaperata, Hydnangium kanuka. The new combinations are Rhodocollybia purpurata and Gerrronema waikanaensis. Mycena stevensoniae is a nom. nov. for Crinipellis roseola, and the new record for New Zealand is Lyophyllum atratum.

A monograph of Crinipellis and Chaetocalathus species growing in tropical Africa is in preparation. During studies of the original material and other specimens, some new taxa were recognized and are described in this paper: Chaetocalathus niduliformis var. bisporus, Crinipellis atrovinosa var. parvispora, C goossensiae, C. minutuloides, C kisanganensis, C. nsimalensis, and C. pseudopalmarum. Crinipellis mauritiensis is described as a nomen provisorum because of the absence of a macroscopic description and the presence of only very poor material. Type revisions of Chaetocalathus niduliformis and Lentinus minutulus caused some taxonomic and nomenclatural novelties that are published here. A new status is proposed for Crinipellis pseudostipitaria var. orientalis.

Crinipellis brunneoaurantiaca, C. pallidibrunnea and C. rubella are described as new species and their taxonomic position is discussed. The two former were collected in subdeciduous tropical forest and the latter in the montane cloud forest, all from the east coast of Mexico (central Veracruz). Crinipellis podocarpi, C. pseudostipitaria var. mesites, C. setipes, recorded in montane cloud forest, and C. tucumanensis, collected in subdeciduous tropical forest, also are discussed. Detailed macro- and microscopic descriptions, illustrations of distinctive microscopic characters and plates are presented for each species.

The present study reports Crinipellis cupreostipes (first described from Thailand) as a new record for Indian mycobiota. A phylogenetic analysis based on nrDNA ITS shows that C. cupreostipes can be readily distinguished from other morphologically similar species such as Crinipellis nigricaulis var. macrospora. A detailed taxonomic description with illustrations and an artificial key to Crinipellis species previously reported from India and its neighboring countries are provided.

A newly isolated alkalitolerant basidiomycetous fungus, identified as Crinipellis sp. RCK-1 was observed to produce laccase. The effect of different physicochemical factors on laccase production was studied. The identification of the important factors (initial pH of the medium, copper and tryptophan) with simple screening experiment involving optimization using single factor at a time strategy, was followed by application of complex response surface design for further maximizing the laccase production and was helpful in defining the effects and interactions of the physiological and nutritional factors. The statistical optimization by response surface methodology resulted in a 27.0-fold (619.9 U ml–1) increase in the production of laccase from Crinipellis sp. RCK-1 when compared to laccase production in unoptimized medium (23.0 U ml–1). The results from the response surface curve suggested that there was interaction between tryptophan and copper in a way that might resulted in positive effect on laccase production from Crinipellis sp. RCK-1. The relatively higher laccase production by Crinipellis sp. RCK-1 showed promise of offering great potential in various biotechnological applications. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Two new species of Agaricales and a new Japanese record for Chaetocalathus fragilis from Ishigaki Island, a southwestern island of Japan, are fully described and illustrated: (1) Crinipellis (section Grisentinae) rhizomorphica sp. nov. produces brownish orange, fibrillose-squamulose basidiomata accompanied by white thread-like rhizomorphs on the dead twig, olivaceous hairs in KOH, and oblong-ellipsoid, relatively long basidiospores; (2) Chaetocalathus (section Holocystis) fragilis is a new record for Japan, growing on the dead twig; (3) Psilocybe (section Cubensae) capitulata sp. nov. forms a furfuraceous-squamulose pileus, cyanescent flesh, a persistent, membranous annulus, capitulate pilocystidia, and has a coprophilous habit on cow dung.

Moniliophthora perniciosa and Moniliophthora roreri are phytopathogenic basidiomycete species that infect cacao causing two important diseases in this crop: "Witches' Broom" and "Frosty Pod Rot", respectively. The ability of species from this genus (Moniliophthora) to cause disease is exceptional in the family Marasmiaceae. Species in closely related genera including, Marasmius, Crinipellis, and Chaetocalathus, are mainly saprotrophs and are not known to cause disease. In this study, the possibility that this phytopathogenic lifestyle has been acquired by horizontal gene transfer (HGT) was investigated. A stringent genome comparison pipeline was used to identify potential genes that have been obtained by Moniliophthora through HGT. This search led to the identification of three genes: a metallo-dependent hydrolase (MDH), a mannitol phosphate dehydrogenase (MPDH), and a family of necrosis-inducing proteins (NEPs). Phylogenetic analysis of these genes suggests that Moniliophthora acquired NEPs from oomycetes, MDH from actinobacteria and MPDH from firmicutes. Based on the known gene functions and on previous studies of M. perniciosa infection and development, a correlation between gene acquisition and the evolution of the phytopathogenic genus Moniliophthora can be postulated.

Physicochemical properties, composition, and structure of the polysaccharides synthesized by the fungi Lentinus edodes, Ganoderma lucidum, and Crinipellis schevchenkovi in a submerged culture were studied. It has been demonstrated that the exo-and endopolysaccharides of submerged mycelia of the fungi studied are similar to the β-1,3-D-glucans of the fruiting bodies.

Four new species of Pouzarella are described from the Dominican Republic and this report is the first of this genus from Hispaniola. A new variety of one of these newly described species is also described from Jamaica. Crinipellis ( Pouzarella ) squamifolia, previously described from Jamaica by Murrill, is also discussed and illustrated. A new record for the Dominican Republic, P. caribaea, is also reported. We now know of seven species and varieties of Pouzarella from the Greater Antillean islands in the Caribbean.

Crinipellis pedemontana, a new agaric growing on stems of dead grasses in an Italian urban park, is described and illustrated. It fits in sect. Grisentinae on account of the green reaction with KOH and ammonia and its bright colored pileus. The unique combination of purple-violaceous tinges in the pileus and the presence of chlamydospores in the pileipellis characterize this species as new. Its taxonomic position within Crinipellis and the related genus Moniliophthora is discussed.

Witches' broom and pod rot are the two most devastating diseases of cocoa in South America and Africa, respectively. Their control by means of phytosanitation and chemical fungicides is labor-intensive, costly and, in many cases, environmentally undesirable. Therefore efforts are made in order to identify alternative, environmentally safe and cost-efficient methods for the control of these pathogens. Promising candidates are components of the neem tree (Azadirachta indica), that have been used for centuries in Asia as insecticides, fungicides, anticonceptionals in popular medicine. Here we report about tests on the effect of various concentrations of extracts from neem leaves on growth of mycelia of Crinipellis and Phytophthora and on germination of spores of Crinipellis. We show a 35% growth reduction of mycelia of Phytophthora on neem leaf extract media, whereas growth of mycelia of Crinipellis was not affected, even at the highest concentration of neem leaf extracts used (35%). However, the most dramatic effect of neem leaf extracts is observed on Crinipellis spore germination, here the extracts (20-35%) reduced germination almost completely. Based on these results, we believe that the neem tree might be a source for the production, on small and medium scale, of an effective and cheap formulation for the control of Crinipellis and Phytophthora.

SUMMARY The basidiomycete Crinipellis perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cocoa). Hypertrophic growth of infected buds ('brooms') is the most dramatic symptom, but the main economic losses derive from pod infection. To identify cocoa genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from infected shoot-tips of the susceptible genotype 'ICS 39' and the resistant 'CAB 214', in both directions. A total of 187 unique sequences were obtained, with 83 from the library enriched for the susceptible 'ICS 39' sequences, and 104 for the resistant 'CAB 214'. By homology search and ontology analyses, the identified sequences were mainly putatively categorized as belonging to 'signal transduction', 'response to biotic and abiotic stress', 'metabolism', 'RNA and DNA metabolism', 'protein metabolism' and 'cellular maintenance' classes. Quantitative reverse transcription amplification (RT-qPCR) of 23 transcripts identified as differentially expressed between genotypes revealed distinct kinetics of gene up-regulation at the asymptomatic stage of the disease. Expression induction in the susceptible 'ICS 39' in response to C. perniciosa was delayed and limited, while in 'CAB 214' there was a quicker and more intense reaction, with two peaks of gene induction at 48 and 120 h after inoculation, corresponding to morphological and biochemical changes previously described during colonization. Similar differences in gene induction were validated for another resistant genotype ('CAB 208') in an independent experiment. Validation of these genes corroborated similar hypothetical mechanisms of resistance described in other pathosystems.

The efficacy of controlling bacterial spot in susceptible tomato plants by using a natural product was compared to a commercial resistance inducer. Plants were sprayed with (a) acibenzolar-S-methyl, ASM [Bion® 50 WG (0.2 g l−1)] and (b) a heterogeneous chitosan suspension (MCp) from Crinipellis perniciosa mycelium. Plants were challenged 4 d later with a virulent strain of Xanthomonas vesicatoria, under greenhouse conditions. In assessing disease, MCp-treated plants showed significant responses, reaching 87% of ASM protection performance against X. vesicatoria leaf spot. Tomato leaves exposed to MCp and ASM were assayed for pathogenesis-related enzymes, lignin deposition and soluble phenolic compounds. Induced resistance (IR) was evidenced by the enhancement of peroxidase (POX), polyphenol oxidase (PPO), and chitinase (CHI) activities at 1–72 h after spraying. These enzymes and phenylalanine ammonia-lyase (PAL) were also observed at 6, 9 and 12 d after spraying (DAS) interval. Treated and inoculated plants showed an increase in lignin deposition. The content of total soluble phenolic compounds decreased significantly by 9 and 12 DAS. The results suggest that (IR) was characterized by increased POX and PPO activities, improving lignification and, to a less extent, by CHI activity.

The aim of this study was to test whether resistance of clones of Theobroma cacao (cocoa) varied between isolates of Moniliophthora (formerly Crinipellis ) perniciosa , the cause of witches’ broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 × Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.

The witches' broom disease caused by the fungus Crinipellis perniciosa is the main phytossanitary constraint for cacao production in Brazil. The integrated management of the disease involves resistance as one of the components. The breeding program conducted by the Brazilian Institution, CEPLAC is directed toward the pyramidation of resistance genes from different sources to achieve a more durable resistance. This study aimed to identify sources of resistance in progenies of cacao accessions collected in the basins of ten Amazonian rivers and compared to progenies from the Peruvian clones 'Scavina 6' and 'Sacavina 12'. Progenies from 40 Amazonian accessions and 'Scavina' were evaluated in the field for six years for witches' broom resistance through multivariate and repeated measurement analyses evaluating the effect of progeny, area, block, year, and their interactions. There were differences in the mean number of vegetative brooms on some Amazonian progenies and 'Scavina' descendants. There was an increase in the number of vegetative brooms in the last year for 'Scavina' progenies, but that was not observed for the Amazonian progenies 64, 66, 156, 194, 195, 269 and 274. There were different gene/alleles for resistance in the Amazonian progenies in comparison to the traditional 'Scavina' accessions. These new sources of resistance will be important for pyramiding resistance genes and consequently increasing the stability and durability of the resistance to witches' broom.

This study investigated the induced defence responses and protective effects on susceptible tomato ( Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ ( Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar- S -methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria , under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.

O objetivo deste trabalho foi avaliar a influência de eliciadores biológicos e químicos sobre as atividades de duas proteínas relacionadas à patogênese (PR), quitinase e beta-1,3-glucanase, em folhas de tomateiro, e avaliar o potencial desses eliciadores na redução do progresso da mancha-foliar causada por Xanthomonas campestris pv. vesicatoria. Plantas de tomateiro da cultivar Santa Cruz Kada foram pulverizadas com: acibenzolar-S-metil (ASM; 0,2 g L-1); formulação biológica proveniente de biomassa cítrica, denominada Ecolife (5 mL L-1); suspensão de quitosana (MCp; 200 g L-1), proveniente de micélio de Crinipellis perniciosa; extrato aquoso de ramos de lobeira (Solanum lycocarpum) infectados por C. perniciosa (VLA; 300 g L-1). As plantas foram desafiadas com um isolado virulento da bactéria, quatro dias depois das pulverizações. Plantas pulverizadas com extratos biológicos mostraram redução da mancha-bacteriana. ASM proporcionou 49,3% de proteção, e foi igual à MCp e Ecolife e superior ao VLA. Este último não diferiu significativamente de MCp e Ecolife. Observou-se maior atividade das duas enzimas nas plantas tratadas, principalmente nas primeiras horas após as pulverizações.

Molecular markers (RAPD, AFLP and microsatellites) were used to generate a linkage map and to identify QTLs associated to witches' broom (Crinipellis perniciosa) resistance in cacao (Theobroma cacao), using 82 individuals of an F2 population derived from the clones ICS-1 (susceptible) and Scavina-6 (resistant). Fifteen evaluations of the number of brooms have been carried out in six years (1997–2002). In order to increase the precision and accuracy in the measures of resistance, each F2 plant was cloned in three replications in a randomized block design with single-tree plots and evaluated over 2 years. Three hundred and forty-two markers were obtained, being 33 microsatellites, 77 AFLPs and 232 RAPDs. The distribution of the number of brooms in the F2 population was skewed to resistance, suggesting the involvement of major genes controlling resistance and the repeatability estimated for resistance was 44%. A strong putative QTL was detected as being related to witches' broom resistance. Associated to this QTL, the microsatellite mTcCIR35 explained 35.5% of the phenotypic variation in resistance. This marker is being used for marker-assisted selection in Scavina-6 progenies, including those selected in private plantations, as an auxiliary tool to the phenotypic selection.

Breaking of hyphae derived from growth of the phytopathogenic fungus Crinipellis perniciosa in liquid media yielded cell aggregates that performed as "quasi single cell" in toxicity assays. When treated with the chemical mutagens 4-nitroquinoleine-1-oxide (4NQO), hydrogen peroxide (H2O2), or paraquat (PAQ) as well as with ultraviolet light (UVC), broken hyphae of C. perniciosa gave a single cell-like response, i.e., survival curves similar to those obtained when treating single-cell suspensions. C. perniciosa had significantly higher UVC resistance than haploid bakers yeast but was more sensitive to 4NQO and extremely sensitive to PAQ and H2O2 when compared to likewise-treated yeast. Haploid C. perniciosa basidiospores (monokaryotic) were significantly more UVC resistant than C. perniciosa broken hyphae and than haploid and diploid yeast wild-type strains. This suggests a high capacity in C. perniciosa for repair of UVC-induced DNA lesions or, alternatively, an efficient protection from UVC irradiation, especially in basidiospores. The pronounced sensitivity of the dikaryotic form of C. perniciosa to PAQ and H2O2 points to a weak protection from oxidative stress-inducing agents.

Witches' broom disease (WBD) of cacao, caused by the hemibiotrophic fungus, Crinipellis perniciosa, exhibits a succession of symptoms that are caused by the biotrophic phase of the fungus. However, the study of this biotrophic phase is limited by its exclusive growth inside the plant or in the presence of callus. Here we report for the first time a method for the growth and maintenance of the biotrophic-like phase of C. perniciosa on a defined medium with metabolites found in the diseased tissues. Our results suggest that glycerol is a key carbon source for this interaction. This is a crucial achievement toward understanding the biology of this fungus during the infectious phase of WBD.

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.

Thevenin, J.-M., Umaharan, R., Surujdeo-Maharaj, S., Latchman, B., Cilas, C., and Butler, D. R. 2005. Relationships between black pod and witches’-broom diseases in Theobroma cacao. Phytopathology 95:13011307. Field observations were conducted from 1998 to 2001 at the International Cocoa Genebank, Trinidad, to evaluate 57 cacao clones for resistance to black pod (BP) and witches’-broom (WB) diseases (caused by Phytophthora sp. and Crinipellis perniciosa, respectively). Each month ripe pods were harvested and the number of healthy and diseased was recorded. The number of brooms on vegetative shoots was recorded three times a year on selected branches. Twenty-three clones showed less than 10% of infection for both BP and WB on pods. Among those, eight clones showed an absence of brooms on the observed branches: IMC 6, MAN 15/60 [BRA], PA 67 [PER], PA 195 [PER], PA 218 [PER], PA 296 [PER], PA 303 [PER], and POUND 32/A [POU]. Broad-sense heritability was estimated at 0.38 and 0.57 for WB disease on pods and shoots, respectively, and at 0.51 for BP disease. Genetic correlation between WB disease on pods and on shoots was low and estimated at 0.39, whereas the correlation between WB and BP diseases on pods was 0.48. To choose putative parents for breeding schemes, it is suggested that clones are first assessed for their level of resistance to WB on shoots, and the most promising individuals are screened for BP with a detached pods test. Further studies are needed to confirm whether the level of resistance to WB on pods can be predicted using an early test on seedlings. Additional keywords: cocoa diseases, field resistance, germ plasm evaluation, polygenic resistance.

This article describes two field trials carried out at La Lola, Costa Rica, to assess control measures against frosty pod rot of cocoa (Theobroma cacao) caused by Moniliophthora (Crinipellis) roreri. In the first, factorial, trial the control agents were applied using motorised mistblowers (MMs) and hydraulic sprayers fitted with a narrow angle cone nozzle. There was an interaction between agents and application methods; together with previous application data for the most active fungicide (copper hydroxide), these trials indicate that best yields are achieved with sprays that maximise deposits on pods. We describe the droplet size spectra produced by a Stihl SR400 MM under a range of conditions because this has become the standard method of fungicide application in this series of trials at La Lola. The factor that had the largest effect on droplet size spectrum was the presence or the absence of a detachable baffle plate in front of the air-shear nozzle. In both trials described here, MMs were fitted with baffle plates, a formulation pump and restrictor transmitting 550 mL min - 1 to deliver an estimated equivalent of 190 L ha - 1 . Copper hydroxide as prophylactic applications at 1500 g a.i. ha - 1 have, to date, shown the most consistent (but incomplete) improvement in healthy pod yield. Use of copper fungicides may be cost effective when farm-gate cocoa prices exceed approximately $1.25 kg - 1 . In these trials, isolates of the hyper-parasitic fungi Clonostachys byssicola and Trichoderma asperellum and two off-patent triazole fungicides (bitertanol and triadimenol) made no significant improvement to healthy yields. The systemic oxathiin fungicide flutolanil, at a dosage of 300 g a.i. ha - 1 , appears to protect pods substantially at early stages but gives proportionately less control of M. roreri than copper at later stages of pod development.

Crinipellis perniciosa causes a serious disease of cacao known as witches’ broom (WB). Heritable resistance to witches’ broom has been used in cacao improvement programs. ‘SCA6’ and ‘SCA12’ are highly resistant and are the most commonly used parents in the breeding schemes. However, SCA hybrids are not resistant to witches’ broom in all production areas. Presumably, different populations of C. perniciosa cause these variable responses. Amplified fragment length polymorphism (AFLP) markers were used to assess variation and population structure in this pathogen. We examined 40 isolates of C. perniciosa and one isolate of Melanotus subcuneiformis. Nine of 64 primer pairs produced consistent and informative DNA amplification, and were used to screen all isolates. Fifteen haplotypes (AFLP fingerprints) were detected with 186 polymorphic markers. Cluster analysis grouped isolates of the C biotype (pathogenic on cacao) from Bolivia, Brazil, Ecuador and Trinidad together in a major cluster that was distinct from isolates of the S biotype (pathogenic on solanaceous hosts) and M. subcuneiformis. Isolates of the C biotype were divided further into well supported, country-specific groups. Segregation of AFLP alleles was not observed among basidiospore isolates from the same basidiome, broom, tree or field, supporting previous reports that the fungus did not outcross. The results corroborated prior conclusions that C. perniciosa was probably introduced into the Bahia state of Brazil from the Amazon basin. Representative isolates from the genetically distinct groups that were revealed will be used to examine pathogenic specialization in C. perniciosa and differential responses that have been reported in ‘SCA6’-derived germplasm.

The basidiomycete fungus Crinipellis perniciosa (Stahel) Singer is the causal agent of Witches' Broom Disease of Cacao (Theobroma cacao L.) which is the main factor limiting cacao production in the Americas. Pod losses of up to 90% are experienced in affected areas as evidenced by the 50% drop in production in Bahia province, Brazil following the arrival of the C. perniciosa in the area in 1989. The disease has proven particularly difficult to control and many farmers in affected areas have given up cacao cultivation. In order to evaluate the potential of endophytes as a biological control agent of this phytopathogen, the endophytic fungal community of resistant and susceptible cacao plants as well as affected branches was studied between 2001 and 2002. The fungal community was identified by morphological traits and rDNA sequencing as belonging to the genera Acremonium, Blastomyces, Botryosphaeria, Cladosporium, Colletotrichum, Cordyceps, Diaporthe, Fusarium, Geotrichum, Gibberella, Gliocladium, Lasiodiplodia, Monilochoetes, Nectria, Pestalotiopsis, Phomopsis, Pleurotus, Pseudofusarium, Rhizopycnis, Syncephalastrum, Trichoderma, Verticillium and Xylaria. These fungi were evaluated both in vitro and in vivo by their ability to inhibit C. perniciosa. Among these, some were identified as potential antagonists, but only one fungus (Gliocladium catenulatum) reduced the incidence of Witches' Broom Disease in cacao seedlings to 70%.

The new species Trichoderma ovalisporum is described and its biocontrol potential against Crinipellis species is analyzed. Using partial nuclear translation elongation factor 1α (EF-1α) and partial nuclear actin gene intron and exon sequences, T. ovalisporum is identified as a member of Trichoderma sect. Trichoderma and as a close relative of T. koningii-like taxa that have ellipsoidal to oblong, smooth conidia. Sequences of the internal transcribed spacer regions 1 and 2 (ITS) of the nuclear ribosomal gene cluster did not resolve the phylogeny of T. ovalisporum and its closest relatives. Trichoderma ovalisporum is morphologically similar to T. koningii, Hypocrea stilbohypoxyli, and three as yet undescribed Trichoderma taxa. It differs from its close relatives in having smaller, ovoidal conidia and in its fast rate of growth at 30 °C. Trichoderma ovalisporum was isolated twice: once from witches’ broom (Crinipellis perniciosa)-infected tissue of a liana (Banisteriopsis caapi, Malpighiaceae) collected in Ecuador. The second isolation was from the healthy bole of a mature tree of Theobroma grandiflorum (cupuacu, Malvaceae) collected in Brazil (Para). The liana isolate reinfected and was reisolated from meristematic tissues of seedlings of Theobroma cacao, and inhibited radial growth of the frosty pod rot pathogen (Crinipellis roreri) in vitro. It also persisted on the surface, and within the tissues, of cocoa pods in the field for at least 10 weeks.

Infection by Crinipellis perniciosa on cacao seedlings prevented the abscission of cotyledons which normally abscise within 32-61 days from seed sowing. The early growth and development of control and artificially inoculated seedlings was investigated for root dry mass (RDM), shoot dry mass (SDM), shoot-to-root ratio (SRR), total dry mass (TDM), cotyledonary dry mass (CDM) and cotyledonary water content (CWC). Cotyledonary reserve mobilization was estimated at 69 and 56% at 5 weeks and 79 and 64% at 8 weeks in control and infected seedlings, respectively. Infection induced the enlargement of tissues but was not accompanied by an increase in dry mass. Infection both delayed and reduced the utilization of cotyledonary food reserves resulting in slower accumulation of RDM, SDM and TDM culminating in a reduction of growth by a factor of 1.6 at 8 weeks. However, the SRR of inoculated plants was similar to that of control plants, suggesting that the balance between the root and shoot systems exists which is unchanged by the effect of the pathogen. CWC was decreased upon senescence to an average of 65% at abscission. The prevention of cotyledonary abscission in infected seedlings is suggested to be due in part to the CDM remaining above a critical level of 20% of the initial CDM. The implication of this response in relation to the infection biology of the pathogen is discussed.

Witches' broom disease, caused by Crinipellis perniciosa, is one of the major fungal diseases causing severe losses to cacao tree (Theobroma cacao L.) plantations in South America. One of the challenges associated with the understanding of the cacao and Crinipellis interaction in genomic studies is the isolation of intact nucleic acids. In this report, we describe a new, successful, and reliable procedure for the isolation of RNA from tissues of cacao tree, both infected and uninfected by Crinipellis. This protocol overcomes the problems associated with the very high amount of polyphenols and polysaccharides present in cacao organs that are not easily removed by conventional extraction procedures. The protocol requires few reagents, uses ultracentrifugation and inexpensive consumables, and can be easily applied in any laboratory. This method produced high-quality RNA that was suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction. We also report the first evidence of RNA isolation from cacao organs infected by C. perniciosa such as meristems and fruits.

The effects of host age, leaf number, host type (clone or seedling), pathogen spore concentration and incubation time on inoculation with Crinipellis perniciosa (witches’ broom disease of cocoa) were studied in greenhouse experiments using susceptible cocoa genotypes. Three methods of inoculation (agar-drop, water-drop and spray) were also tested. An optimized inoculation method was selected and tested for its repeatability as well as its ability to discriminate between various levels of resistance to C. perniciosa in cocoa. The optimized method (350 000 viable basidiospores per mL, 60 h incubation, agar-drop technique) produced 100% infection repeatedly, on both clonal and seedling plants of a susceptible genotype. Seedling age (2–12 months) and leaf number did not significantly affect the percentage of plants with symptoms or broom characteristics. This method discriminated effectively between the various levels of resistance in 14 cocoa genotypes and is recommended as an inoculation method to identify levels of resistance in germplasm collections. Symptom severity was shown to be a better measure of resistance than infection success.

Morphological, cytological and molecular evidence is presented which confirms that the frosty pod rot pathogen of cocoa, formerly classified as the mitosporic fungus Moniliophthora roreri (Deuteromycota), belongs to the hymenomycetous genus Crinipellis (Basidiomycota) and that two varieties should now be recognized: Crinipellis roreri var. roreri and the new variety C. roreri var. gileri. The latter was collected on Theobroma gileri, an endemic tree of submontane forests in north-west Ecuador, and can be distinguished from Ecuadorian and Peruvian isolates from cocoa (T. cacao) on the basis of spore morphology, incompatibility and nucleotide sequence data. As with var. roreri, meiosis is shown to occur within the dispersive and infective spore stage of var. gileri and these meiospores are interpreted to represent a much modified probasidium. In addition, in a field inoculation experiment, an isolate from T. gileri proved to be noninfective to cocoa pods when compared with positive control strains isolated from T. cacao in western Ecuador and T. bicolor in eastern Ecuador. It is concluded that var. gileri is the vestigial progenitor of the frosty pod rot pathogen of cocoa, with a host range and distribution restricted to T. gileri in the mesic forests of north-west South America.