Fungistatic and fungicidal effects of the ionophores monensin and tetronasin on the rumen fungus Neocallimastix sp. LM1

The ionophore antibiotics monensin and tetronasin have been reported to inhibit anaerobic fungi in vitro, and are suitable for animal use. In this study, their effectiveness in removing the anaerobic fungus Neocallimastix sp. LM1 from the rumen was investigated in vitro. Both antibiotics were fungistatic: tetronasin at 0.5 microgram/ml and monensin at 1.0 microgram/ml; exposure for 24 h did not inhibit subsequent growth after removal of the ionophore. The ionophores were fungicidal at much higher concentrations, 1 microgram/ml for tetronasin and 16 micrograms/ml for monensin. It seems likely that the combination of relatively high inhibitory dose and the fungistatic nature of monensin would explain difficulties in using this compound to eliminate anaerobic fungi from the rumens of experimental animals.     

In vitro antifungal activities of anidulafungin and micafungin, licensed agents and the investigational triazole posaconazole as determined by NCCLS methods for 12,052 fungal isolates: review of the literature

The echinocandins anidulafungin and micafungin and the triazole posaconazole are currently undergoing phase III clinical trials. Caspofungin and voriconazole have recently been licensed for the treatment of aspergillosis (both agents), other less common mould (voriconazole) and candidal (caspofungin) infections. This review summarizes the published in vitro data obtained by NCCLS or NCCLS modified methods on the in vitro fungistatic and fungicidal activities of these five agents for yeasts and moulds in comparison to the established agents, amphotericin B, fluconazole, itraconazole, and flucytosine. Among the yeasts, the echinocandins have less activity for Candida parapsilosis and Candida guilliermondii, no activity for Cryptococcus neoformans and Trichosporon spp., but good fungistatic and fungicidal activity in vivo and in vitro for most of the other Candida spp.; this fungicidal activity has been reported by minimum fungicidal concentrations (MFCs) or time kill curve results. The new triazoles exhibit good fungistatic activity (but not fungicidal) for most Candida spp., C. neoformans, and Trichosporon spp. For the Aspergillus spp. evaluated, the echinocandins have similar or better fungistatic activity than those of amphotericin B and the triazoles, but fungicidal activity has been demonstrated only with amphotericin B and the triazoles, with the exception of fluconazole. Most studies showed posaconazole and voriconazole minimum inhibitory concentrations (MICs) ranging from 0.25 to 8 microg/ml for non-solani Fusarium spp., while MIC and minimum effective concentration (MEC) endpoints of the echinocandins were >8 microg/ml. The fungistatic activity of the triazoles is also superior to that of the echinocandins for most of the dimorphic fungi and the Zygomycetes. However, micafungin has activity for the mould phase of most dimorphic fungi, but not for the parasitic or yeast phase of Paracoccidioides brasiliensis. The echinocandins appear to have variable and species dependent fungistatic activity for the dematiaceous fungi, but all agents have poor or no activity against most isolates of Scedosporium prolificans. Only amphotericin B exhibit good fungistatic activity against the Zygomycetes. The combination of caspofungin with some triazoles, amphotericin B or liposomal amphotericin B has been synergistic in vitro, in animal models and in patients. Breakpoints are not available for any mould and antifungal agent combination. In vitro/in vivo correlations should aid in the interpretation of these results, but standard testing conditions are needed for the echinocandins, especially for mould testing, to obtain reliable results.     

Acromyrmex octospinosus (Hymenoptera: Formicidae) management: effects of TRAMILs fungicidal plant extracts

Leaf-cutting ants, Acromyrmex octospinosus (Reich), are considering among the most important pest species of the New World. Until now, the main insecticides used for controlling these ants were synthetic chemicals. Leaf-cutting ants live in obligate symbiosis with abasidiomycete fungus, Leucocoprinus gongylophorus (Heim) Moeller. The crucial role of this symbiotic partner in the nest of leaf-cutting ants has prompted us to focus on A. octospinosus management through the use of fungicides in our study. Five parts of plants identified for their antifungal potential through TRAMIL ethnopharmacological surveys were tested: 1) bulbs of Allium cepa L.; 2) seed pods of Allium sativum L.; 3) green fruits of Lycopersicon esculentum L.; 4) leaves of Manihot esculenta Crantz; and 5) leaves of Senna alata (L.) Roxburgh. One plant extract with strong fungicidal activity (S. alata) against L. gongylophorus was found. The other extracts had lesser fungistatic or fungicidal effects depending on the concentrations used. The data presented in this study showed that TRAMILs fungicidal plant extracts have potential to control the symbiotic fungus of leaf cutting ants, in particular a foliage extract of S. alata.     

Biosynthesis of jasmonic acid in a plant pathogenic fungus, Lasiodiplodia theobromae

Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosynthesis in this fungus has yet to be explored. Administration of [1-(13)C] and [2-(13)C] NaOAc into L. theobromae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of 12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC-MS analysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L. theobromae. (2)H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of [9,10,12,13,15,16-(2)H(6)] linolenic acid showed that five deuterium atoms remained intact. In plants, this is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed stereoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing in the facial selectivity of the enone reduction.     

Fungicidal activity of essential oils of Cinnamomum zeylanicum (L.) and Syzygium aromaticum (L.) Merr et L.M. Perry against crown rot and anthracnose pathogens isolated from banana

AIMS: To develop a post-harvest treatment system against post-harvest fungal pathogens of banana using natural products. METHODS AND RESULTS: Colletotrichum musae was isolated and identified as the causative agent responsible for anthracnose peel blemishes while three fungi, namely Lasiodiplodia theobromae, C. musae and Fusarium proliferatum, were identified as causative agents responsible for crown rot. During the liquid bioassay, cinnamon [Cinnamomum zeylanicum (L.)] leaf, bark and clove [Syzygium aromaticum (L.)] oils were tested against the anthracnose and crown rot pathogens. The test oils were fungistatic and fungicidal against the test pathogens within a range of 0.03-0.11% (v/v). CONCLUSIONS: Cinnamon and clove essential oils could be used as antifungal agents to manage post harvest fungal diseases of banana. SIGNIFICANCE AND IMPACT OF THE STUDY: Cinnamon and clove essential oil could be used as alternative post-harvest treatments on banana. Banana treated with essential oil is chemically safe and acceptable to consumers. Benomyl (Benlate), which is currently used to manage fungal pathogens, can cause adverse health effects and could be replaced with volatile essential oils.     

Occurrence of Dermatomycoses and In-Vitro Therapeutic Efficacy of Three Antifungal Drugs on the Growth of Epidermophyton Floccosum

The occurrence of dermatomycoses and the in-vitro therapeutic efficacy of some antifungal agents on dermatomycotic organisms were investigated. Of the 550 primary school children screened, the incidence was one hundred (18%), 70 were males (representing 20% of the males screened) and 30 females (15% of the females sampled). The differences between male and female prevalence were insignificant. Three species of dermatophytes were isolated and identified. These were Microsporum canis, Trichophyton tonsurans and Epidermophyton floccosum. The antifungal agents tested on E. floccosum were griseofulvin, terbinafine and ketoconazole. They produced different sized zones of inhibition against the growth of E. floccosum. Griseofulvin exhibited a 50% inhibition of growth on E. floccosum at 63.00 mg/L. Terbinafine on the other hand exhibited varying levels of inhibition of growth at varying concentrations, at 0.07 mg/L, terbinafine achieved 46% inhibition of growth on E. floccosum. The drug achieved 100% inhibition of growth on the isolate at 61.81 mg/L. In the case of ketoconazole, 50% inhibition of growth was achieved at 100 mg/L while 100% inhibition of growth was achieved at 200 mg/L. The antifungal effects of the three drugs were confirmed by broth dilution tests where terbinafine was found to be fungistatic on the growth of E. floccosum at concentrations ranging from 0.013-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. Ketoconazole was found to inhibit the growth of E. floccosum at 0.003-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. It however did not succeed in killing the isolate under the same range of concentrations. Griseofulvin exhibited fungistatic effects on the growth of E. floccusum at 0.013-1.700 mg/L. In conclusion, ketoconazole and griseofulvin were found to be fungistatic and not fungicidal while terbinafine was both fungistatic and fungicidal on the pathogen. Terbinafine was found to be the most effective drug in inhibiting E. floccosum.     

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.     

Antimicrobial activity of [2-(methacryloyloxy)ethyl]trimethylammonium chloride against Candida spp

BackgroundCandida-associated denture stomatitis is the most common manifestation of oral candidal infection, caused mainly by Candida albicans. Several authors have attempted to add antifungal agents or antiseptics to denture temporary soft lining materials or to denture acrylic resins, without relevant results. Therefore, the investigation of a quaternary ammonium functionalized compound [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MADQUAT), which copolymerizes with methacrylates and which could act as a fungal inhibitor, is of paramount importance.AimsTo evaluate the in vitro activity of MADQUAT against Candida species.MethodsThirty-one Candida strains were used to determine the in vitro antifungal activity of this compound. The minimum inhibitory concentrations and minimum fungicidal concentrations of MADQUAT and nystatin were determined.ResultsMADQUAT showed antifungal properties at concentrations of 6.25 to > 100 mg/ml, and fungicidal activity between 25 and > 100 mg/ml. The quantitative determinations of the fungistatic and fungicidal activity of MADQUAT showed fungistatic activity against all Candida albicans, Candida krusei and Candida parapsilosis strains, revealing fungicidal activity against some strains of the other species.ConclusionsMADQUAT has antifungal activity against Candida spp. Moreover, the sensitivity to this substance varies across the different species in terms of MIC values and fungicidal or fungistatic activity.     

INVESTIGATIONS ON FUNGICIDES

Twenty (aryloxythio)trichloromethanes were examined for in vitro fungicidal activity against six fungi. All compounds showed a direct fungistatic effect and some exhibited a marked fumigant action. Good protectant action against Alternaria solani on tomato and Botrytis fabae on broad bean was obtained with certain compounds, but none was better than the three standard protectants used for comparison. When supplied to plants through their roots, eight conferred significant systemic fungicidal protection against Alternaria solani in tomato, but there was no significant protection against Botrytis fabae in broad bean. In preliminary tests the 2:4:5-trichlorophenoxythio analogue gave promising results when examined as a fumigant fungicide for eradication of downy mildew in lettuce plants, for reducing lenticel rot in stored apples and for preventing blue-mould in oranges.     

Quantitative characterization of antagonistic activity of lactobacilli

In this work the method of serial dilutions of lactobacilli in two-layer agar was used. On the agar surface bacterial or fungal cultures were applied at different time intervals. A special quantitative characteristic was introduced. L. plantarum strain 8P-A3 was shown to have the maximum antagonistic activity. In great amounts L. casei and L. reuteri are capable to suppress the growth of bacteria and fungi. All lactobacilli under study produced a pronounced bactericidal effect on Pseudomonas, had different influence on the viability of Escherichia and staphylococci and exhibited fungistatic and fungicidal action only when inoculated at high concentrations.     

一种袋栽黑木耳共生菌的鉴定及其共生效应初步研究

【目的】研究袋栽黑木耳某种共生菌对黑木耳生长的影响。【方法】经分离纯化,以及形态学和分子系统学分析,鉴定该真菌的分类地位;通过不同时间接种共生菌到黑木耳菌棒的方法,研究两者的共生效应;通过子实体营养成分分析,研究共生菌对黑木耳子实体营养结构的影响。【结果】经分离纯化获得菌株B-1,形态学和分子系统学分析将该菌株鉴定为毛色二孢属的可可毛色二孢(Lasiodiplodia theobromae)。通过不同时间接种黑木耳菌棒发现,此真菌与黑木耳间存在偏性共生关系。它对于黑木耳是偏利共生,还是偏害共生取决于黑木耳菌丝在菌棒中是否具有先占优势。黑木耳子实体营养成分分析显示,可可毛色二孢的存在改变了黑木耳子实体中营养结构,但黑木耳子实体形态特征无明显变化。【结论】可可毛色二孢与袋栽黑木耳存在着偏性共生关系,该现象为首次报道。     

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl₃ on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 °C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 M of FeCl₃ at both apotransferrin concentrations. By contrast, addition of FeCl₃ to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl₃. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.     

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C₆ to C₁₃ were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C₁₁) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C₁₀) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C₁₂) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H⁺-ATPase. Octanol (C₈) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C₉–C₁₂) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.     

Broth Microdilution and Time–Kill Testing of Caspofungin, Voriconazole, Amphotericin B and their Combinations Against Clinical Isolates of Candida krusei

Treatment of invasive Candida krusei infections can be difficult due to its intrinsic fluconazole resistance and its reduced susceptibility to amphotericin B and flucytosine. Caspofungin (CAS) acts on a different cellular target, and its combination with voriconazole (VOR) or amphotericin B (AmB) appears promising. We evaluated the activity of CAS, VOR and AmB alone and in combination at 1/4, 1, 4xMIC concentrations by time–kill method against 30 C. krusei isolates. All isolates were susceptible to CAS and VOR; AmB MICs were 2 μg/ml for 50% of isolates by broth microdilution. CAS showed a fast killing activity at all concentrations; it was fungistatic at 1/4xMICs and fungicidal at 1-4xMICs in general. VOR displayed a concentration-independent fungistatic activity against all isolates. AmB exhibited a concentration-dependent activity; it was fungistatic at 1/4-1xMIC and fungicidal at 4xMIC. The most common interaction was indifference for both combinations. Frequency of synergic interaction for the VOR + CAS combination was 66.7% at 1/4xMIC after 48 h. The best results for CAS + AmB combination were obtained at 4xMIC in the first 4–8 h; synergic interaction was detected for 20 isolates (66.7%) at 4xMIC after 4 h. Consequently, VOR and CAS alone have been found effective, and high AmB MICs are remarkable against clinical C. krusei isolates in vitro. The combinations of CAS with VOR or AmB have exhibited promising results.     

Lasiodiplodia theobromae Keratitis: A Case Report and Review of Literature

A case report and review of literature is reported of a rare case of fungal keratitis from eastern India. A 32-year-old woman with a history of vegetative trauma presented with keratitis in left eye. Microbiological examination of corneal scraping showed refractile hyphae with aseptate branching filaments and black pigmented colonies on multiple solid agar medium. Organism was identified from culture using D1/D2 region of LSU (Large Sub Unit: 28S rDNA)-based molecular technique. PCR amplified a band with a sequence that was 100?% homologous with Lasiodiplodia theobromae. The organism was susceptible to amphotericin B and voriconazole and demonstrated resistance to itraconazole and fluconazole. A therapeutic keratoplasty was performed following non-responsiveness to initial topical voriconazole (2?%) therapy. Recurrence in graft was controlled with topical voriconazole and intracameral amphotericin B. However, the graft failed at the end of 3?months. L. theobromae is a rare cause of fungal keratitis. Management of these cases is difficult, often involving surgical procedures.     

In vitro activity of fluconazole and amphotericin B against Candida inconspicua clinical isolates as determined by the time-kill method

Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.     

Culture filtrate of Lasiodiplodia theobromae restricts the development of natural resistance in Brassica nigra plants

Culture filtrate of Lasiodiplodia theobromae increased respiration rate, phenylalanine ammonia lyase activity, and levels of hydrogen peroxide, lipid peroxides and salicylic acid in B. nigra plants. Salicylic acid (SA) level increased for 1 hr of interaction and reduced later. Development of systemic acquired resistance (SAR) was found restricted in plants infected with L. theobromae due to deficiency of SA, which is a major signal for development of SAR. Exogenously supplied SA did develop resistance and plant death was delayed. It was hypothesized that deficiency of SA could be due to jasmonic acid produced by fungus that inhibits SA biosynthesis.     

Fungistatic activity of the sternal gland secretion of the dampwood termite <Emphasis Type="Italic">Zootermopsis angusticollis</Emphasis>

Summary In vivo and in vitro studies indicate that cuticular chemicals from the ventral region of the abdomen where the sternal gland of the dampwood termite Zootermopsis angusticollis is located have fungistatic properties. Germination rates of conidia of the entomopathogenic fungus Metarhizium anisopliae were significantly reduced from 91% (controls) to 38.5% after nymphs walked over conidia-seeded agar medium, but did not differ from controls when the sternal gland and surrounding cuticle were sealed with nail polish. In vitro studies show that germination of fungal conidia was also significantly reduced following incubation with cuticular extracts of either sternal or tergal segments suggesting that cuticular exudates in general may have antifungal properties. Extracts of sternites had greater fungistatic activity than extracts of tergites, but the difference was not statistically significant. Extracts of the sternal gland significantly reduced germination rates by up to 9%. Germination rates were significantly reduced when conidia were incubated with n-hexanoic acid, or its vapor. n-Hexanoic acid has been recovered from whole body extracts of Zootermopsis nevadensis and may indeed be a component of the sternal gland of Z. angusticollis. Here we suggest that sternal gland secretions in termites may have had the original function of controlling microbes within the nest and their prominent role in communication may have evolved secondarily.Received 18 April 2003; revised 20 November and 17 December 2003; accepted 19 January 2004.     

Evaluation of some fungicides for seed treatment and foliar application in management of damping-off of seedlings and blight of rapeseed caused by Alternaria brassicae

Eighteen fungicides were first evaluated for their effects on growth of Alternaria brassicae and for ascertaining their fungicidal and fungistatic natures in artificial cultures. The chemicals emerging fungicidal in action, were later evaluated for their efficacy as seed treatment and foliar application in the management of damping-off of seedlings and blight of rapeseed separately. Of 18 fungicides tested, six fungicides, viz., Dithane M-45, Dithane Z-78, Ziram, Difolatan-80, Blitox-50 and Benlate completely inhibited the growth of the pathogen and were fungicidal in action. Thiram and Brestan-60, which also caused total growth inhibition, were, however, fungistatic. Benlate (0.1 %) followed by Dithane M-45 was best seed-dressing fungicide for controlling damping-off of seedlings. Dithane M-45 (0.2%) followed by Dithane Z-78 as foliar spray was most effective for controlling the blight and increasing the yield in field trials.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.