Molecular characterisation of fungal endophytic morphospecies associated with the indigenous forest tree, Theobroma gileri, in Ecuador

Fungal endophytes were isolated from healthy stems and pods of Theobroma gileri, an alternative host of the frosty pod rot pathogen of cacao. Non-sporulating isolates were grouped into 46 different morphological species according to their colony morphology. Many of these morphospecies were assumed to be basidiomycetes and, therefore, were of particular interest. Basidiomycetous endophytes have received far less attention than ascomycetes and also have potential as biological control agents of the basidiomycetous pathogens of T. cacao: Moniliophthora roreri (frosty pod rot pathogen) and M. perniciosa (witches' broom disease). The morphospecies were further characterised by molecular analyses. Amplification of the nuLSU was undertaken for phylogenetic placement of these non-sporulating cultures and revealed a total of 31 different taxa of which 15 were basidiomycetes belonging to the class Agaricomycetes, and 16 ascomycetes primarily belonging to the Sordariomycetes.     

Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America

Trichoderma theobromicola and T. paucisporum spp. nov. are described. Trichoderma theobromicola was isolated as an endophyte from the trunk of a healthy cacao tree (Theobroma cacao, Malvaceae) in Amazonian Peru; it sporulates profusely on common mycological media. Trichoderma paucisporum is represented by two cultures that were obtained in Ecuador from cacao pods partially infected with frosty pod rot, Moniliophthora roreri; it sporulates sporadically and most cultures remain sterile on common media and autoclaved rice. It sporulates more reliably on synthetic low-nutrient agar (SNA) but produces few conidia. Trichoderma theobromicola was reintroduced into cacao seedlings through shoot inoculation and was recovered from stems but not from leaves, indicating that it is an endophytic species. Both produced a volatile/diffusable antibiotic that inhibited development of M. roreri in vitro and on-pod trials. Neither species demonstrated significant direct in vitro mycoparasitic activity against M. roreri.     

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species

Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Selecting Bacterial Strains for Use in the Biocontrol of Diseases Caused by <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">Alternaria alternata</Emphasis> in Sweet Pepper Plants

More than 500 isolates of bacteria were obtained from the aerial part and rhizosphere of sweet pepper (Capsicum annuum L.) plants harvested from different places in the Region of Murcia (Spain). The isolates were purified and assayed in vitro against Phytophthora capsici and Alternaria alternata. Sixty isolates (12 %) produced an inhibition zone against at least one of the pathogens, while ten had a strongly inhibitory effect on both pathogens assayed. Microscopic observation of interactions zone showed cell vacuolisation, hyphae lysis and spilling of cytoplasm content of the pathogens in the culture media. These ten isolates were then chosen for biocontrol of Phytophthora root rot and Alternaria leaf spots of pepper plants in vivo. Four of them denominated HS93, LS234, LS523 and LS674 reduced P. capsici root rot by 80, 51, 49 and 54 %, respectively, and A. alternata leaf spots by 54, 74, 62 and 53 %. HS93 belongs to the genus Bacillus and probably the species subtilis, while LS234, LS523 and LS674 belong to the genus Bacillus and probably the species licheniformis. Dry mass of plants treated with these bacteria was significantly higher than that of non-treated and inoculated plants.     

Geographical and Temporal Changes of Foliar Fungal Endophytes Associated with the Invasive Plant Ageratina adenophora

Endophytes may gradually accumulate in the new geographic range of a non-native plant, just as pathogens do. To test this hypothesis, the dynamics of colonization and diversity of foliar fungal endophytes of non-native Ageratina adenophora were investigated. Previous reports showed that the time since the initial introduction (1930s) of A. adenophora into China varied among populations. Endophytes were sampled in three provinces of Southwest China in 21 sites that varied from 20 to 70 years since the introduction of A. adenophora from its native Central America. Endophyte isolation frequencies varied from 1.87 % to 60.23 % overall in a total of 4,032 leaf fragments. Based on ITS sequence variations, 463 fungal endophytes were distinguished as 112 operational taxonomic units (OTUs) belonging to the Sordariomycetes (77 OTUs, 373 isolates), Dothideomycetes (18 OTUs, 38 isolates), and Agaricomycetes (17 OTUs, 52 strains) classes. Colletotrichum (28.51 %), Nemania (14.90 %), Phomopsis (13.17 %), and Xylaria (4.97 %) were the most abundant genera. Both endophyte diversity and overall isolation frequency increased with time since introduction. The genetic differentiation of the fungus Colletotrichum gloeosporioides indicated that the dispersal of endophytes was likely affected by a combination of geographic factors and the invasion history of the host A. adenophora.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Suppression of gray leaf spot (blast) of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate

Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10⁸ cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf.     

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.     

Fungal endophyte diversity and community patterns in healthy and yellowing leaves of Citrus limon

Fungal endophytes on citrus plants have been little studied, and the effects of citrus diseases on their incidence and diversity have not been addressed. In this study, we examined the foliar fungal endophytes of Citrus limon in the vicinity of Yaoundé, Cameroon, with emphasis on the differences between endophyte communities in healthy and yellowing leaves. From 82.3 % of the 480 leaf fragments, a total of 482 isolates were recovered and analysis of ITS sequences revealed 20 phylotypes. All fungal endophytes were ascomycetes and, except for one species, were common plant pathogens. Mycosphaerella and its anamorphs (34.2 % of all isolates), and Colletotrichum gloeosporioides (50.4 % of all isolates), were isolated most frequently. Mycosphaerellaceous species dominated in healthy leaves, and were absent from yellowing leaves. C. gloeosporioides was isolated significantly more frequently from yellowing than healthy leaves. Yellowing leaves had a significantly higher overall infection frequency but, in contrast, the least species diversity. Difference in the endophyte assemblages of healthy and yellowing leaves suggests that yellowing of leaves may facilitate the incidence of certain endophytes and impose growth inhibition on others.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Entomopathogenic fungi (Hypocreales) for control of potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in an area endemic for zebra chip disease of potato

Potato psyllid, Bactericera cockerelli, is a serious pest of potato and other solanaceous vegetables in the United States, Mexico, Central America, and New Zealand and is responsible for transmission of Candidatus Liberibacter solanacearum which causes a disease known as “zebra chip” (ZC). Entomopathogenic fungi could provide a viable component for an integrated pest management strategy for control of B. cockerelli and other potato pest insects. Three field trials of commercial formulations of Metarhizium anisopliae (F 52®, Novozymes Biologicals) and Isaria fumosorosea (Pfr 97®, Certis USA) and abamectin (Agri-Mek®, Syngenta, USA) were conducted in Weslaco, Texas. Rates are expressed in quantity of product delivered in 375–470 l of water/ha. F 52 applied at 0.51, 1.1, and 2.2 l/ha and Agri-Mek applied at 584 ml/ha produced reductions of B. cockerelli eggs and nymphs of 45%, 59%, 67%, and 63%, respectively. Only Agri-Mek significantly reduced plant damage. Pfr 97 at 1.1 kg/ha with and without 1% Trilogy® (neem oil, Certis, USA), and Agri-Mek at 584 ml/ha resulted in psyllid reductions of 78%, 76%, and 84%, respectively. Significantly decreased plant damage and ZC symptoms were observed for all treatments. Tuber yields for Pfr plus Trilogy and Agri-Mek were significantly higher than the control. F 52 applied at 1.1 and 2.2 l/ha and Pfr 97 at 1.1 and 2.2 kg/ha produced 62%, 62%, 66%, and 65% reduction, respectively. Tuber yield for both rates of Pfr and the high rate of F 52 were significantly higher than the control. All fungal treatments significantly reduced plant damage and ZC symptoms.     

Selection of Trichoderma stromaticum isolates for efficient biological control of witches’ broom disease in cacao

Witches’ broom is the most devastating disease of cacao in Brazil, and losses to it entail serious socio-economical and environmental problems. Biological control of the causal agent Moniliophthora perniciosa (Mp) using the antagonistic fungus Trichoderma stromaticum (Ts) is promising, although the identification of superior isolates is necessary. Here, we report a study on the selection of more effective Ts isolates based on field experiments. Sixty-three Ts isolates from a local collection were applied on brooms and placed under typical conditions of shaded-cacao plantations in southeastern Bahia State (Brazil), during two periods of three months each. The percentages of Ts sporulation and incidence and severity of Mp were the parameters used for biocontrol assessments. The results from both experiments were very distinct, indicating a high phenotypic variation in this collection and suggesting a significant effect of the environment in the Ts–Mp interaction. Ts-sporulation rates were negatively correlated with the presence of Mp in the brooms and a number of isolates reduced Mp incidence more efficiently than the reference isolate. Contrasting isolates in their efficiency of reducing Mp incidence were selected and further tested in four subsequent field trials for validation purposes. The results partially confirmed their biocontrol phenotypes but also suggested isolate-specific responses to environmental variations. Inhibition of Mp-basidiospore germination by total protein secreted in culture supernatants of Ts isolates correlated well with field results and revealed a potentially useful procedure for pre-screening of large collections towards selection of better biological control isolates. The characteristics and efficiency of the method as a reliable protocol for identification of superior BCAs in the witches’ broom—cacao pathosystem is discussed.     

Physiological characteristics of several rumen protozoa grown in vitro with observations on within and among species variation

When fed equal amounts of substrate, two Epidinium caudatum clone cultures of markedly different size produced similar volumes of microbial protoplasm. Addition of up to 50% volume of 72 h culture medium had no inhibitory effects on growth of Epidinium. Two clone cultures of Epidinium caudatum from Australia had longer generation times and showed less substrate attachment when compared to Ohio clones of this same species. Substitution of alfalfa for orchardgrass in the normal substrate increased Epidinium concentrations, while feeding only ground orchardgrass or alfalfa resulted in a marked decrease or disappearance of the protozoa. Eudiplodinium impalae, isolated from rumen contents of a steer in Australia, was successfully cultured, with generation times for this species averaging 11.3 h. Reducing particle size of the substrates by ball-milling was detrimental for growth of Entodinium and Epidinium; however, Eudiplodinium increased in concentration. Significant concentration differences were observed among six clone cultures of Epidinium obtained from Europe. A generation time of 18.7 h was measured for Enoploplastron triloricatum when the culture was transferred every 12 h. Lowering the incubation temperature to 34 °C completely inhibited protozoal growth of Epidinium and Entodinium exiguum after 12 days, but not for Entodinium caudatum.     

Comparison of the diversity,composition, and host recurrence of xylariaceous endophytes in subtropical,cool temperate,and subboreal regions in Japan

The diversity, composition, and host recurrence of endophytic fungi in the Xylariaceae were compared in subtropical (ST), cool temperate (CT), and subboreal forests (SB) in Japan based on the 28S ribosomal DNA sequences from fungal isolates. A total of 610 isolates were obtained from the leaves of 167 tree species in three sites, which were classified into 42 operational taxonomic units (OTUs) at the 99 % similarity level of the 28S rDNA sequence. ST, CT, and SB yielded 31, 13, and three OTUs, respectively. The OTU richness, diversity, and evenness of fungal communities were in the order: ST > CT > SB. The 42 OTUs were assigned to nine genera in the Xylariaceae: Xylaria, Annulohypoxylon, Anthostomella, Biscogniauxia, Nemania, Hypoxylon, Muscodor, Daldinia, and Rosellinia. Xylarioid isolates in the subfamily Xylarioideae outnumbered Hypoxyloid isolates in the subfamily Hypoxyloideae in ST and CT, whereas the opposite was found in SB. Sørensen’s quotient of similarity was generally low between the three sites. Host recurrence of fungal OTUs was evaluated with the degree of specialization of interaction network between xylariaceous endophytes and plant species and compared between the three sites. We found that the networks in the three sites showed a significantly higher degree of specialization than simulated networks, where partners were associated randomly. Permutational multivariate analyses of variance indicated that plant family and leaf trait significantly affected the OTU composition in ST, which can account for the specialization of interaction network and host recurrence of xylariaceous endophytes.     

Molecular characterization of Echinococcus isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina

Human cystic echinococcosis is a highly endemic zoonotic disease in the province of Neuquén, Patagonia Argentina, although a hydatid control programme has been carried out since 1970. Human infection due to Echinococcus canadensis (G6 genotype) is frequent in Neuquén. However, the reservoir for this species remains undetermined in a region where camels are absent. We investigated the fertility, viability and molecular epidemiology of hydatid cysts obtained from local goats, pigs and sheep in order to identify the possible reservoirs of E. canadensis (G6). We also analyzed isolates from infected dogs. A total of 67 isolates were identified by the DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Cysts from sheep (n = 16), goats (n = 23) and pigs (n = 18) and adult worms from 10 infected dogs were analyzed. The fertility of the hydatid cysts was 78.6%; 90.4% and 94.4% for sheep, goats and pigs, respectively. We detected E. canadensis (G6) in 21 of 23 goat samples and in 1 dog isolate, E. canadensis (G7) in all the pig isolates, E. granulosus sensu stricto (G3) in 1 sheep and the G1 genotype in 15 sheep, 2 goats and 9 dog samples. The G1 haplotypes included the common sheep strain sequence and 2 microvariants of this sequence. E. granulosus sensu stricto (G3) is described for the first time in South America. We conclude that goats act as reservoir for E. canadensis (G6) in Neuquén, and that control strategies may have to be adapted to local molecular epidemiology to improve the control of parasite transmission.