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This datasheet on Iguana iguana covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Biology & Ecology, Natural Enemies, Impacts, Uses, Prevention/Control, Management, Further Information.

The Lesser Antilles, in the Eastern Caribbean, is inhabited by threeIguanaspecies: the Lesser Antillean iguanaIguana delicatissima, which is endemic to the northernmost islands of the Lesser Antilles, the introduced common iguana from South America,Iguana iguana iguana, represented also by the two newly described endemic subspeciesIguana iguana sanctaluciaefrom Saint Lucia andIguana iguana insularisfrom Saint Vincent and the Grenadines, and Grenada, and the introducedIguana rhinolophafrom Central America. Drawing on both morphological and genetic data, this paper describes theIguanapopulations from Saba and Montserrat as a new species,Iguana melanoderma. This species is recognized on the basis of the following combination of characteristics: private microsatellite alleles, unique mitochondrial ND4 haplotypes, a distinctive black spot between the eye and tympanum, a dorsal carpet pattern on juveniles and young adults, a darkening of body coloration with aging (except for the anterior part of the snout), a black dewlap, pink on the jowl, the high number of large tubercular nape scales, fewer than ten medium sized–triangular dewlap spikes, high dorsal spikes, and lack of horns on the snout. This new melanistic taxon is threatened by unsustainable harvesting (including for the pet trade) and both competition and hybridization from escaped or released invasive alien iguanas (I. iguana iguanaandI. rhinolopha) from South and Central America, respectively. The authors call for action to conserveIguana melanodermain Saba and Montserrat and for further research to investigate its relationship to other melanistic iguanas from the Virgin Islands and coastal islands of Venezuela.

The Lesser Antilles, in the Eastern Caribbean, were long considered to have only two species in the genus Iguana Laurenti 1768: the Lesser Antillean iguana Iguana delicatissima, which is endemic to parts of the Lesser Antilles, and the Common green iguana Iguana iguana, which also occurs throughout Central and South America. No subspecies are currently recognised. However, herpetologists and reptile collectors have pointed out strong physical differences between some of the island populations of Iguana iguana and those from the continent. Drawing on both morphological and genetic data, this paper describes two subspecies of the Common green iguana Iguana iguana from the southern Lesser Antilles, specifically the countries of Saint Lucia Iguana iguana sanctaluciae and Iguana iguana insularis from St Vincent & the Grenadines, and Grenada. The form on the island of Saint Vincent has not been identified. The new subspecies are described based on the following unique combination of characters: Presence of high median and medium to small lateral horns on the snout; Small subtympanic plate not exceeding 20% of the eardrum size; Two or three scales of decreasing size anterior to the subtympanic plate; Fewer than ten small to medium triangular gular spikes; Medium sized dewlap; Low number of small to medium dispersed nuchal tubercles; Dark brown iris, with the white of the eye visible; Oval, prominent nostril; Short and relatively flat head; High dorsal spines; No swelling of the jowls in reproductively active males. Iguana iguana sanctaluciae has in adults vertical black stripes on body and tail and a black dewlap whereas Iguana iguana insularis is pale grey or creamy white in adults. Both subspecies are globally threatened by unsustainable hunting (including the pet trade) and by invasive alien species, including hybridization from invasive iguanas from South and Central America (I. iguana iguana and I. rhinolopha, considered here as full species) that have become established in all three countries. The authors call for stronger measures to conserve the remaining purebred Iguana i. insularis and Iguana i. sanctaluciae ssp. nov. throughout their ranges and for further research to identify other cryptic species and subspecies of Iguana in the Lesser Antilles.

The Lesser Antilles, in the Eastern Caribbean, were long considered to have only two species in the genusIguanaLaurenti 1768: the Lesser Antillean iguanaIguana delicatissima, which is endemic to parts of the Lesser Antilles, and the common green iguanaIguana iguana, which also occurs throughout Central and South America. However, herpetologists and reptile collectors have pointed out strong physical differences between some of the island populations ofIguana iguanaand those from the continent. Drawing on both morphological and genetic data, this paper describes a third speciesIguana insularissp. nov. from the southern Lesser Antilles, specifically the countries of Saint Lucia, St Vincent & the Grenadines, and Grenada. The new species is described based on the following unique combination of characters: Presence of high median and medium to small lateral horns on the snout; Small subtympanic plate not exceeding 20% of the eardrum size; Two or three scales of decreasing size anterior to the subtympanic plate; Fewer than ten small to medium triangular gular spikes; Medium sized dewlap; Low number of small to medium dispersed nuchal tubercles; Dark brown iris, with the white of the eye visible; Oval, prominent nostril; Short and relatively flat head; High dorsal spines; No swelling of the jowls in reproductively active males; Colour of head, body and tail changing from green to pale grey or creamy white in old adults; Vertical black stripes on body and tail, fading with age in some populations. This paper furthermore distinguishes two subspecies:Iguana insularis insularisfrom the Grenada Bank (comprising Grenada and the Grenadine islands), andIguana insularis sanctaluciaefrom Saint Lucia. The form on the island of Saint Vincent has not been identified. Both subspecies are globally threatened by unsustainable hunting (including the pet trade) and by invasive alien species, including hybridization from invasive iguanas from South and Central America (I. iguanaandI. rhinolopha, considered here as full species) that have become established in all three countries. The authors call for stronger measures to conserve the remaining purebredIguana insularissp. nov. throughout its range and for further research to identify other cryptic species and subspecies ofIguanain the Lesser Antilles.

Pharmaceutical Research, 21 (1), ISSN:0724-8741, ISSN:1573-904X

Cocoa (Theobroma cacao) pod diseases cause tremendous losses, frequently eliminating over 80% of potential yield. Biological control has previously shown promise to supplement cultural control methods practiced by the majority of smallholders. Mixed Clonostachys inocula were most effective against multiple pathogens in Peru, but not in Costa Rica, where Clonostachys spp. seemed to antagonize Trichoderma spp. The objective of our study was to systematically investigate the mycoparasitic interactions between Clonostachys and Trichoderma species, in order to enable the design of mixed inocula in a rational manner. Specifically, we aimed to quantify dominance of one mycoparasite genus over the other using in vitro tests as well as field experiments. The compatibility of Peruvian and Costa Rican isolates of known efficacy was tested by pair-wise confrontations on water agar as well as two host-range assays, one of which mycoparasites were offered another mycoparasite as only host and one where conidia of a Clonostachys and a Trichoderma isolate were mixed at ratios ranging from 1:100 to 100:1 in order to determine dominance on the susceptible host, Phytophthora palmivora. A field study quantified survival and establishment of selected mixtures on surface-sterilized cocoa pods. Hyphal interactions and host-range studies coincided in identifying Clonostachys rosea as the most aggressive mycoparasite, closely followed by C. byssicola. Trichoderma spp. were least aggressive; instead, they were highly susceptible to Clonostachys spp. The outcome of interactions depended on the species used and occasionally even on the isolates confronting each other. The country of origin, however, was irrelevant. Parasitic growth of C. rosea was unaffected by Trichoderma harzianum, even at an inoculum ratio of 1:100. Parasitic growth of Trichoderma spp. progressively slowed as C. rosea concentrations rose, but the effect varied in strength with different isolates. When C. rosea and Trichoderma sp. were applied to cocoa pods in the field either separately or in combination at a ratio of 5:1, C. rosea dominated at the expense of Trichoderma sp., which was suppressed. At equal or ten times higher concentrations of T. harzianum, C. rosea was temporarily suppressed, but from two weeks after application, remained the dominant and persistent pod colonizer. Native Trichoderma spp. were poor pioneer colonizers of surface-sterilized pods and applied populations vanished within less than four weeks. Our results suggest that, although Clonostachys spp. antagonize Trichoderma spp., the incompatibility can be partly overcome by adjusting the inoculum ratio of mixed inocula in favor of Trichoderma spp. Therefore, mycoparasitism among several promising biocontrol candidates do not necessarily rule out their combination into a mixed inoculum, but can be addressed by formulating susceptible isolates at higher inoculum ratios. This, however, will not sustainably improve surface coverage by poorly colonizing Trichoderma spp. Therefore, optimal strains for the habitat and task are needed first.

To aid the development of compatible biocontrol inocula, a prescreening method for the prediction of compatibility of fungal antagonists was developed. Compatibility between 18 Clonostachys isolates with known antagonistic capabilities against Phytophthora palmivora was tested using intra- or interisolate pairings (dual cultures) on water agar plates, a hyphal interaction experiment and a modified double host-range experiment. Almost all inter- or intraisolate pairings of Clonostachys isolates showed growth inhibition zones and did not show free hyphal intermingling. A hyphal interaction experiment on water agar demonstrated that the aggressiveness of a Clonostachys isolate and its susceptibility to mycopar- asitism were unrelated phenomena. However the level of aggressiveness and/or susceptibility of an isolate were largely dependant on the isolate with which it was challenged. The degree of growth-inhibition caused by an isolate was unrelated to the hyphal damaged it caused or received. In the double host-range experi- ment all possible pairs from four Clonostachys isolates were inoculated in different ratios (10 000-fold range) on plates precolonized with one of two P. palmivora isolates. The results showed that antagonistic capabil- ities of certain combinations were affected by the Clonostachys isolates. The primary host, P. palmivora, did not affect antagonistic capabilities; whereas inocu- lum ratio did. Of note, it was not possible to predict the outcome of the double host range on the basis of the results of the hyphal interaction experiment. In conclusion the competitive abilities of Clonostachys isolates depend on the partner with which they are applied and less on resource availability. The double host-range test as developed here might provide the most representative tool to date to test compatibility of fungal antagonists to be used in biocontrol inocula. However the link between the results of the double host-range test and field efficacy of biocontrol inocula remains to be investigated.

Frosty pod rot (FPR), caused by Moniliophthora roreri, reduces yields of cocoa (Theobroma cacao) by over 80% within a few years of disease outbreak. Both biological and chemical control approaches are being developed to supplement cultural management. Our objectives were to optimize the agent(s), their formulation and application regime for FPR control in Costa Rica. In order to economize scarce resources for field research, we opted for an iterative optimization of treatments. We aimed to (partly) replace copper hydroxide of proven efficacy with two systemic agents, the coevolved endophyte Trichoderma ovalisporum and the oxathiin flutolanil, and assessed four stickers for improving the agents' performance. Over 3 years, we selected effective agents and their combinations: eight of 18 treatments increased yields. Orthogonal contrasts extracted three general trends: (1) Copper should be applied in water, whereas the systemic agents were best formulated with a sticker. There was no difference between the stickers BreakThru and NP-7. (2) If the first rule was observed, a switch from systemic agent to the contact fungicide in mid season was beneficial. The optimum timing for this change requires further definition. (3) T. ovalisporum-containing agents controlled black pod better than flutolanil-based ones; both systemic agents were equally effective in controlling the more severe FPR. We discuss potential benefits of switching agents in the context of organic agriculture and argue that coevolved endophytes may be particularly suited for the classical biocontrol of Moniliophthora spp. in cocoa planted outside its South American centre of origin.

This chapter discusses the advances in biological control of cacao diseases over the last 15 years. Most attention has been focused on biological control of frosty pod rot (Moniliophthora roreri), witches’ broom (Moniliophthora perniciosa) and black pod disease (Phytophthora spp.). Research on biocontrol of other diseases in the cacao phyllosphere or rhizosphere is scarce or in its infancy. There is, however, a steady increase in information regarding the factors influencing and the mechanisms underlying biological control of cacao diseases as well as practical aspects such as inoculum production, formulation and application. There has been a clear shift away from inundative approaches using epiphytic BCAs towards more classical biocontrol approaches using bacterial and fungal endophytes as well as vesicular arbuscular mycorrhiza. These have the advantage that they can permanently establish themselves in the cacao tree. Moreover, besides direct competition for space and nutrients, antibiosis and mycoparasitism, through induced resistance and growth promotion, endophytes have a larger arsenal of mechanisms through which they can help protect their host. Endophytic BCAs could thus provide more effective and sustainable disease control. Recent advances in our understanding of the mechanisms through which endophytic biocontrol agents can reduce pest and disease impact provide possibilities for innovative disease control strategies, including combination therapies together with natural or chemical products. Continued work on production, formulation and application is also necessary in order for biocontrol to become economically interesting. However, biological control will not become a stand-alone solution for disease control but should become part of integrated pest management strategies, with cultural management as a central and reinforcing pillar.

In this chapter, we consider some of the “root-infecting” pathogens of Theobroma cacao. Despite sometimes being described as minor pathogens of the crop, they can cause serious losses locally. We have chosen to examine four pathogens—Verticillium dahliae (Verticillium wilt or sudden death), Rigidoporus microporus (white root disease), Rosellinia spp. (black rot), and Phellinus noxious (brown root rot). These are all soil-borne, root-infecting fungi, but whilst the basidiomycetes and Rosellinia (ascomycete) are unspecialized, opportunistic root rot pathogens whose pathogenicity is part of a saprophytic lifestyle, Verticillium is a highly specialized pathogen adapted for colonization of the xylem system. Yet, despite differences in their pathogenicity, there are some important practical similarities with regard to their management. These pathogens are extremely difficult to manage given their soil-borne nature, colonization of alternative hosts and/or colonization of woody debris, and the perennial nature of the cacao host plant. Currently, cultural methods are often used as the main management approaches with chemical control being considered too costly or ineffective. Development of breeding programs against the unspecialized pathogens is one area of research that should be expanded; successful breeding programs have been undertaken for Verticillium in Brazil. Biological control is another area where further work is needed for all these pathogens both with regard to the direct application of biological control agents (BCAs) themselves and on the interaction with BCAs and various soil amendments to enhance naturally occurring antagonists. There may be differences in approach here based on modes of pathogenicity, but inevitably, we should be aiming at an integrated approach for these pathogens.

Frosty pod rot (FPR), caused by Moniliophthora roreri, and black pod (BP), caused by Phytophthora spp., of cocoa (Theobroma cacao) cause combined pod losses of more than 80% in Panama. Biological control of both diseases appeared promising in Peru and is desired by certified organic producers in Panama. We evaluated both local and Peruvian fungal antagonists in participatory trials on smallholdings during two complete production cycles. Furthermore, we tested the influence of a 3% v/v cane molasses amendment on biocontrol efficacy, yield and population dynamics of mycoparasites on the cocoa pod. Significant variation was observed between the two years: FPR was more severe in the first year, BP in the second. FPR was significantly reduced by biocontrol agents (BCAs), but not by the molasses amendment. However, BCAs responded differently to molasses in both years. All BCAs reduced inoculum production by M. roreri with no consistent effect of molasses. BCAs had a lesser and more variable effect on BP, whereas molasses reduced BP slightly by increasing the efficacy of native antagonists. All BCAs and the molasses amendment enhanced the percentage of healthy pods. Molasses was beneficial to absolute yield, but only one inoculum improved yield significantly in the first year. Populations of a Peruvian Trichoderma asperellum isolate remained high for over two months after application to surface-sterilized pods. Molasses had no effect on establishment or survival of this antagonist or recolonization by any native mycoparasite. The reasons for enhanced biocontrol efficacy of the molasses formulation requires further investigation.

Rosellinia species have been recorded all over the world. Many species occur as saprobes and only a few are well-known root pathogens. Although Rosellinia spp. have been recognized for more than 130 years to cause losses in many economically important crops and trees, the few existing sets of quantitative data have not previously been collated. Our objective is to integrate available knowledge on the biology and control of Rosellinia root rots, with emphasis on the tropics and subtropics, i.e. root rots caused by Rosellinia bunodes , R. necatrix and R. pepo. The review will summarize briefly the taxonomy, geographical distribution, ecology, host-range, disease symptoms and spread of Rosellinia in order to provide a rational basis for the choice of control options. Different avenues explored to control Rosellinia are presented: cultural control, solarization, genetic resistance, chemical and biological control. Successful Rosellinia control usually consists of an integrated approach, invariably centring around cultural control options. In the short term, chemical control may be best suited to supplement cultural measures, while medium and long term solutions could be provided by biological control and genetic resistance, respectively, if prioritized research needs are addressed satisfactorily.

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous α-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial andenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.

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.

Human calcitonin and its C-terminal fragment 9–32 (hCT(9–32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9–32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9–32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9–32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9–32) in solution, transforming to α-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9–32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9–32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9–32).

Biocontrol agents of numerous insect pests and fungal pathogens exist but virtually nothing is known about their interaction if used simultaneously. Our objective was to investigate the compatibility of the entomopathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus, and the broad host-range mycoparasites Clonostachys spp., Trichoderma harzianum and Lecanicillium lecanii. In vitro host-range tests revealed that M. anisopliae was highly susceptible to all mycoparasites tested. B. bassiana was attacked by Clonostachys rosea, and P. fumosoroseus. was resistant to mycoparasites. M. anisopliae but not P. fumosoroseus killed nymphs of Bemisia tabaci in bioassays. B. bassiana and M. anisopliae proved lethal to Cosmopolites sordidus, Diatraea saccharalis and Sitophilus oryzae. Coapplication of mycoparasites with entomopathogens did not affect their biocontrol efficacy in vivo, although the reisolation success of entomopathogens could be significantly reduced, especially from smaller insect species. Trichoderma spp. were reisolated from mycoparasite-treated insects more frequently than C. rosea. The coapplication of the highly susceptible M. anisopliae generally enhanced mycoparasite recovery. Mycofungicide preparations caused some insect mortality but less than a copper hydroxide fungicide which is still permissible in organic agriculture. We concluded that the tested entomopathogens and mycoparasites are compatible elements of integrated pest management.

Many small (temporary) collections of fungi maintained by plant pathologists during their research receive inadequate attention to ensure stability. Maintaining collections of fungi in pure and viable conditions, minimising physiological and morphological changes is, however, a necessity. The objective of this study was to find preservation techniques for three Rosellinia isolates used in our plant pathogenic research. Various inert and nutritious carriers, solid as well as liquid, were used to test their suitability for conserving these Rosellinia isolates. Different cryoprotectants, cooling rates and thawing rates were tested to optimise liquid nitrogen storage procedures. Survival and/or growth rate were assessed over time. Rosellinia bunodes was the most difficult to store with survival not exceeding six to nine months using traditional storage methods in mineral oil and silica gel. Storage of Rosellinia necatrix and Rosellinia pepo was successful for periods up to at least 16 months in several carriers and for up to two years for R. necatrix in silica gel. Storage in liquid nitrogen proved no problem for R. necatrix or R. pepo with a 100% survival in all cases, although radial growth rates after recuperation were affected by cryoprotectant and thawing rates. Storage of R. bunodes was more difficult and survival as well as growth rates were affected by cryoprotectant and thawing rates. Cooling rates did not affect radial growth in any of the isolates. The results showed that development of a generalised procedure for storage of our Rosellinia species was not possible and successful storage protocols had to be developed for individual isolates.

Gene therapy still awaits a broader application, since safe and efficient gene delivery is a major problem. Also for the investigation of signal transduction and intracellular trafficking, delivery systems for hydrophilic macromolecules that are easy to use are needed. Several peptide-based delivery systems have been developed during the last years. We present here a novel carrier peptide derived from human calcitonin that is capable of transfecting human neuroblastoma cells by complex formation with a plasmid. Because of the peptide's physiological origin, cytotoxic effects are not expected.

Baker, C. J., Harrington, T. C., Krauss, U., and Alfenas, A. C. 2003. Genetic variability and host specialization in the Latin American clade of Ceratocystis fimbriata. Phytopathology 93:1274-1284. The Ceratocystis fimbriata complex includes many undescribed species that cause wilt and canker diseases of many economically important plants. Phylogenetic analyses of DNA sequences have delineated three geographic clades within Ceratocystis fimbriata. This study examined host specialization in the Latin American clade, in which a number of lineages were identified using sequences of the internal transcribed spacer (ITS) region of the rDNA. Three host-associated lineages were identified from cacao (Theobroma cacao), sweet potato (Ipomoea batatas), and sycamore (Platanus spp.), respectively. Isolates from these three lineages showed strong host specialization in reciprocal inoculation experiments on these three hosts. Six cacao isolates from Ecuador, Trinidad, and Columbia differed genetically from other cacao isolates and were not pathogenic to cacao in inoculation tests. Further evidence of host specialization within the Latin American clade of Ceratocystis fimbriata was demonstrated in inoculation experiments in growth chambers using sweet potato, sycamore, Colocasia esculenta, coffee (Coffea arabica), and mango (Mangifera indica) plants; inoculation experiments in Brazil using Brazilian isolates from cacao, Eucalyptus spp., mango, and Gmelina arborea; and inoculation experiments in Costa Rica using Costa Rican isolates from cacao, coffee, and Xanthosoma sp. Hosts native to the Americas appeared to be colonized by only select pathogen genotypes, whereas nonnative hosts were colonized by several genotypes. We hypothesize that local populations of Ceratocystis fimbriata have specialized to different hosts; some of these populations are nascent species, and some host-specialized genotypes have been moved to new areas by humans.

Rosellinia spp. cause substantial mortality of cocoa (Theobroma cacao), especially in acidic soils rich in organic matter (O.M.) and, reportedly, low available phosphate. Cultural and chemical control are relatively ineffective and a supplementary biocontrol strategy would be highly desirable. Our aim was the isolation and evaluation of promising mycoparasites against Rosellinia of cocoa. We isolated 45 mycoparasites using a baiting technique. Three of them and a commercial isolate were prioritized for further evaluation based on their broad hostrange and/or effectiveness in pot bioassays. Because Rosellinia is favored by the same soil factors as mycoparasites, we investigated the effect of soil pH (range 4–6), O.M. (6–12%), and phosphate (0– 1000 mg kg −1 ) on their interaction. In vitro, both pathogens and antagonists grew well on a wide range of phosphate (0– 9500 mg kg −1 ) and at various pH (3.0–10.0) levels. The pH optimum usually coincided with the soil of origin and the phosphate optimum depended on the phosphate source used. In pot bioassays using natural soils, mixtures of antagonists exhibited more control than single strains and mixtures were effective over a wider range of soil conditions. High O.M. favored the pathogen more than its biocontrol agents. Soil pH interacted with the treatments such that biocontrol was most efficient at higher pH values and with the highest mixtures of antagonists. The addition of phosphate enhanced plant growth but had little effect on biocontrol efficacy. We conclude that mixtures of mycoparasites should be field tested in combination with liming and removal of O.M.

Severe and often therapy-limiting side effects are a major obstacle in cancer chemotherapy. New delivery concepts reducing systemic side effects are needed in order to optimize anticancer therapies. Several approaches have been followed, most of them concentrating on macromolecular carriers like liposomes, monoclonal antibodies, serum proteins or polyethylene glycol. We present here a novel type of anthracycline conjugate, using a small carrier peptide derived from the peptide hormone human calcitonin (hCT). The carrier peptide hCT(9-32) has so far been shown to be capable of transporting fluorophores or proteins across cellular membranes. Two different carrier peptide-daunorubicin conjugates were prepared, one with an acid-stable amide bond, the second with an acid-labile hydrazone bond. In vitro studies with daunorubicin linked to the carrier peptide via an acid-labile hydrazone bond demonstrated comparable cytotoxicity to daunorubicin in various daunorubicin sensitive cell lines (neuroblastoma cell lines SK-N-MC and SMS-KAN; HEK 293 T cells). In addition, fluorescence microscopy provided further insight into the mechanism of uptake of the carrier peptide hCT(9-32), indicating that endosomal compartments with reduced pH are involved in the intracellular release of daunorubicin.

A model of the field dynamics of Moniliophthora roreri, its management and economics was developed to evaluate net returns of various management strategies to farmers in Central America. The model is primarily based on the cropping patterns and economics of cocoa production in Costa Rica. With minimal inputs the model can be adapted to simulate the crop phenology and economics in any country, where the disease causes significant economic loss. The model is a time-based deterministic spreadsheet model that simulates the production and management of one hectare of cocoa at weekly intervals over a user-selected two-year period. The model demonstrates the need for frequent stripping of infected pods to prevent sporulating pods accumulating in the field, under a broad range of economic scenarios. The model allows the user to evaluate potential extension advice in terms of user-definable “real world” variables including: frequency of harvesting and stripping of infected pods; losses of ripe unharvested pods to rodents; ability to identify infected pods; international cocoa prices; farm-gate prices and premiums; and labour costs and requirements.

Three native and two commercial biocontrol agents (Clonostachys rosea and Trichoderma spp.) were evaluated against the cocoa diseases moniliasis, witches’ broom, and black pod. Antagonists were applied either separately or as mixed inoculum in comparison with a copper fungicide and a nontreated control. Cultural control (weekly removal of diseased pods) was practiced in all treatments. Field trials were conducted on neglected cocoa farms in eastern Peru from 1998 to 2000. The fungicide treatment and C. rosea strain G-4 did not reduce disease. The other single-strain antagonists reduced moniliasis. Additionally, Trichoderma longibrachiatum and Trichoderma stromaticum reduced witches’ broom and Trichoderma virens reduced black pod. With strain mixtures, yield increases of up to 15% were obtained. A mixture of four antagonists was superior to mixtures of two or three antagonists with respect to multiple disease control and yield. Biocontrol in combination with cultural control was more economical than cultural control alone; chemical control was least economical. Fertilization improved yields by 11% independent of the disease control measure and compensated for the additional costs with net returns improving by 9%. Increasing application frequencies of a mixed biocontrol inoculum improved moniliasis and witches’ broom control linearly. Moniliasis exhibited a stronger response. Witches’ broom was significantly lower than the nontreated control only if ten applications were administered in two-week intervals. This application frequency increased yields by 15%. It was followed by three applications adjusted to the production cycle (yield increase: 12%). Three adjusted applications were the most economical biocontrol strategy under the conditions of eastern Peru. Net returns were increased by 12%. Recommendations for technology transfer are presented. 2002 Elsevier Science (USA). All rights reserved.

Five native mycoparasitic strains of Clonostachys rosea and three of Trichoderma spp. were isolated from healthy cocoa tissue or basidiocarps of Crinipellis perniciosa using a baiting technique. They were compared singly or in combination with the commercial biocontrol agent Trichoderma virens (SoilGuard) for their potential to control three cocoa pod diseases: moniliasis, caused by Moniliophthora roreri; witches' broom, caused by C. perniciosa; and black pod, caused by Phytophthora palmivora. All isolates except Trichoderma T-1 inhibited basidiocarp formation of C. perniciosa under controlled conditions. The remaining isolates except Trichoderma T-3 reduced vegetative broom formation in a seedling bioassay. Clonostachys rosea G-3 and Trichoderma strains T-2 and T-3 significantly reduced symptoms caused by M. roreri in a seedling bioassay. Host-range studies identified P. palmivora as most susceptible to mycoparasitism and M. roreri as most resistant; C. perniciosa was intermediate. Different degrees of susceptibility were discovered at the pathogen strain level, especially for P. palmivora. However, broad host-range mycoparasites which attacked all three pathogen species were identified. Under field conditions, all selected treatments except a combination of C. rosea G-2 + G-3 reduced moniliasis, the main disease, significantly by 14.6–24.9% as compared with optimized, cultural control alone. No significant reduction of witches' broom or black pod was achieved but a combination of five C. rosea strains (G-1, G-2, G-3, G-4 + G-5) performed consistently best against all three diseases simultaneously. Yield increased by 16.7% and net returns by 24%. Control of moniliasis and yield were positively correlated to the number of mycoparasites in the inoculum. The results suggest that simultaneous biocontrol of the three major cocoa pod diseases with mycoparasite mixtures is highly promising. Future development strategies are discussed.

This review gives an account of control approaches used against the crown rot disease complex of banana in the Windward Islands during the past 20 yr. Fungicide-impregnated pads which were unique to the islands and offered advantages to the smallholder production system were replaced by a mini wet-pack in which bananas are dip-treated in a fungicide suspension, a procedure which was less error-prone. Neither method effected consistent disease control and was acceptable in terms of worker safety and environmental protection. Several regional R&D activities towards improved and environmentally friendly crown rot control are presented. They include cultural methods, physical methods, mode of fungicide application, natural chemicals as alternatives to synthetic fungicides and biological control.

Thirty mycoparasites were detected using plates precolonized with pathogens of the crown-rot disease complex: Colletotrichum musae (6 strains), Fusarium moniliforme var. subglutinans (2 strains), F. moniliforme, Fusarium pallidoroseum, Botryodiplodia theobromae , and Nigrospora sphaerica (1 strain each). Dry leaves, dry flower residues, green leaves, and green peel were compared as mycoparasite sources. Dry leaves were the most prolific source, whereas green material collected in the canopy yielded the least number of isolates. Host species differed significantly in their suitability as bait for mycoparasites. C. musae was the best host for the isolation of most mycoparasites; fusaria were least colonized. No difference between strains of the same host species was detected during isolation. In contrast, in dual culture, different C. musae strains exhibited significant differences in susceptibility to mycoparasitism. The consequence of this finding for practical biocontrol is discussed. Thirteen aggressive antagonists, including Pythium sp., Trichoderma sp., Gliocladium sp., and Verticillium sp., were selected for further evaluation. Preliminary efficacy, host range, aggressiveness against survival structures (conidia and haustoria), and tolerance to fungicides presently used for crown-rot control were investigated. Eight mycoparasites had a reasonable number of desirable traits, but no single strain exhibited all features required for consistent biocontrol. It is suggested that future studies should consider combining two or three strains and/or to apply them with a reduced rate of a fungicide combined into the inoculum.

Two assessment methods for crown rot were compared: the widely used, categorical 0–5 scale and depth of rot penetration. Both were very highly correlated; the latter was more sensitive. The development of a bioassay for testing for crown rot pathogens and control methods is described. The rotting of green peel disks dipped in spore suspensions was significantly correlated with crown rot progress of whole clusters for both highly pathogenic C. musae and weakly pathogenic F. moniliforme isolates using inoculum concentrations covering a range of five orders of magnitude. The bioassay is specific to crown rot pathogens and detects efficient control measures. Because it is reliable, simple and rapid, it is suggested as a routine pre-screen for biocontrol agents against crown rot. The steps required for the establishment of a representative bioassay are explained and the final, optimised practical protocol is described.

Axenic cultures of peanut (Arachis hypogaea L.) roots can be initiated from in vitro cultured embryos and shoot meristems. Embryo axes produced more shoots than tissue taken from axillary meristems. For tissue derived from shoot cultures, Virginia cultivars had a higher percentage of rooting explants than a Spanish cultivar. Inoculation with Agrobacterium rhizogenes increased root yields. However, root propagation in liquid medium was unsuccessful. On the other hand, the use of detached leaves, incubated on a sand/mineral liquid medium, led to vigorous root production after inoculation with A. rhizogenes, regardless of the bacterial strain used. These roots could be propagated subsequently in liquid medium. The advantages of the detached leaf technique are discussed.

We assessed the effect of calcium-based anti-stress and fertilization products oncocoa diseases, yield and quality. Calcium levels in cocoa seeds were elevated by up to 25% w/w. Cocoa bean defects, pod index, pH and organoleptic parameters were unaffected by treatments. Impaction did not improve yield (average 179.6kgha-1yr-1), but led to a reduction of cherelle wilt from 3.7% to 1.6% over two seasons. Impaction withSpeedo CaNincreased yield by over 40%, from 190.7kgha-1yr-1to 273.7kgha-1yr-1, by stimulating pod production. Diseases, most notably frosty pod rot, claimed over 80% of pods and were unaffected by the above products. Thus, we combined them with the biocontrol agentTrichoderma ovalisporum. The agents proved to be compatible and augmented the number of healthy pods harvested, but not as much as a copper fungicide.&nbsp

A pimaricin-resistant strain of Mucor hiemalis Wehmer was applied as spores to the hypocotyls of groundnut ( Arachis hypogaea L.) and to non-planted soil 12 days after sowing in field conditions in Malawi. Its subsequent distribution in the root zone was followed by selective reisolation over 51 days and related to measured rainfall and soil physical characteristics. The fungus spread to a maximum recorded depth of 58cm on the tap root, much further than any previous reported distance for water-infiltration of fungal spores in soil. Transport in soil adjacent to tap roots and detection at depth on root laterals was much less (max. 22 and 9 cm, respectively) and detectable movement was negligible in non-planted soil. Mechanisms of spore transport on roots are suggested and some previously proposed factors are reinterpreted. The findings indicate considerable potential to manipulate the rhizosphere microbial populations by seed inoculation under tropical climatic conditions.

We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47–57) and penetratin(43–58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin–Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9–32) was through reversed-phase HPLC fractionation and peak allocation by MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry) or direct MALDI–TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9–32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9–32), may be used as a template to suggest structural modifications towards improved CPP performance.

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide–cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32).

Pharmaceutical Research, 21 (7), ISSN:0724-8741, ISSN:1573-904X

This paper describes a factorial trial designed to investigate the delivery systems of fungal biological agents (Clonostachys rosea), applied as single strain or as strain mixture, in comparison with copper hydroxide as a chemical standard against Moniliophthora roreri in cocoa. Application techniques compared were motorised mistblowers fitted with rotary atomisers and hydraulic sprayers fitted with cone nozzles giving a narrow angle of spray. The third factor was the presence or absence of an emulsifiable rape-seed adjuvant oil in the tank mixture. Copper fungicide was the best agent for moniliasis control and resulted in the highest yield. Both the mycofungicides reduced sporulation of the pathogen. Net reduction of inoculum was best for the hydraulic sprayer-applied copper fungicide and the motorised mistblower-applied single-strain biocontrol agent. Overall, directional hydraulic sprays were found to be somewhat superior to the motorised mistblower technique, with its dissipated cone of spray. Addition of the adjuvant oil was the least important of the factors, but efficacy depended on the agent used, with a significant enhancement in the efficacy of copper with oil but a detrimental effect on C. rosea.

Mycoparasites collected from aerial parts of the cocoa plant ( Theobroma cacao ) have shown great promise in the control of black pod, caused by Phytophthora palmivora , and moniliasis, caused by Moniliophthora roreri . However, the ecology of epiphytic mycoparasites is still poorly understood although it has a direct bearing on applied biocontrol practices, ranging from the identification and isolation of promising biocontrol candidates to formulation needs and required application frequency. One objective of this study was to determine the natural abundance of mycoparasites on cocoa flowers and pods in relation to crop development stage and cultivar. For this purpose, native mycoparasites were detected on cocoa flowers and pods using the precolonised plate baiting technique. Furthermore, the survival of an applied Clonostachys rosea isolate on cocoa pods on shaded and non-shaded trees was compared as well as the recolonisation patterns of surface-sterilised pods by native mycoparasites under these conditions. Clonostachys spp. were the most commonly isolated native mycoparasites, followed by Fusarium spp. No differences in the occurrence of native, epiphytic mycoparasites were observed between the three main cocoa cultivars, ‘Criollo’, ‘Forastero’ and ‘Trinitario’, nor between clones within these groups. Thus, a single biocontrol inoculum can be suitable for application to cultivar mixtures of cocoa commonly grown together in a field. Different susceptibility classes of segregating F1 populations of hybrids with resistance against M. roreri and P. palmivora supported similar population levels and taxonomic assemblages of mycoparasites. Therefore, we reject the hypothesis that these antagonists mediate resistance. Mycoparasite abundance and genetic disease resistance to black pod and moniliasis are independent phenomena and should lead to additive effects if employed simultaneously in an integrated disease management programme. The survival of applied C. rosea was not affected by the shading regime or any other meteorological parameter measured. On the other hand, recolonisation of surface-sterilised cocoa pods by most native mycoparasites was faster in the shade. Only Trichoderma spp. colonised pods exposed to direct sunlight faster than shaded ones. The implications for the design of biocontrol inocula and formulation technology are discussed.

Carrier peptides offer new opportunities to overcome problems in cellular drug delivery. Their objectives are improved cellular uptake or permeation of biological membranes, which are important pharmacokinetic features for the cellular distribution of therapeutics. Previously, human calcitonin (hCT) and selected C-terminal hCT fragments have been shown to be internalized and to permeate the epithelium of the nasal mucosa. To assess the potential of hCT-derived carrier peptides for cellular internalization of a model protein we fused enhanced green fluorescent protein (EGFP) and the [C(8)]hCT8-32 fragment by using expressed protein ligation (EPL). EGFP thioester was obtained by intein-mediated purification with an affinity chitin-binding tag (the IMPACT system, based on protein splicing). Internalization of EGFP-[C(8)]hCT8-32 by excised bovine nasal mucosa was monitored by confocal laser scanning microscopy. This novel conjugate displayed internalization into some sectors of the mucosa, whereas EGFP itself was not capable of translocation. Thus, we demonstrate successful internalization of a model protein through ligation to an hCT-derived carrier peptide, which has potential for the delivery of therapeutics. At this point the respective mechanism of translocation is unknown.

Economic mass production is a crucial stage in the development of biocontrol agents. This paper compares different solid substrates with and without preceding liquid culture for efficacy and economics in the scaled-up production of the mycoparasitic biocontrol agent Clonostachys rosea. In a basic liquid medium (BLM) containing (in g l−1): molasses (80), neopeptone (10) and yeast extract (2), Clonostachys rosea produced 107 conidia ml−1 after 4 d in agitated culture. Spore viability was 99% and did not differ from that of conidia derived from potato dextrose agar plates. Guata, an inert polyester fibre, coated with BLM and cassava starch (30 g l−1), was the best solid matrix yielding 109 conidia g−1 after 20 d. On a volume basis after extraction and on a production unit basis, Guata was in a similar range to Biodac®, a granular cellulose complex, which had also been enriched with BLM, producing up to 107 conidia ml−1 or over 1010 conidia per 23 × 28 cm polypropylene bag, the production unit in which all solid matrices were incubated. Rice was the lowest-yielding solid matrix regardless of quality. Chlamydospores were not observed in liquid or on solid media. All conidia produced on solid matrices had a viability >90%. Conidia produced on Biodac had a higher viability (95.6%) than spores produced on Guata (93.1%) after 24 h on potato-dextrose plates. However, production on Biodac was more variable and negatively affected by prefermentation in liquid culture, whereas production on Guata and rice was unaffected by the origin of the starting inoculum. Guata of medium thickness (1.27 cm) was superior to thicker (2.54 cm) or thinner (0.64 cm) sheets with respect to both yield per gram and per production unit. Although prefermentation neither improved yield nor reduced cost, it increased throughput by a factor five and may be advantageous where fermentor and/or incubator space is limited. Biodac and Guata were the most economical solid matrices; inoculum to treat one hectare (3 × 1012 conidia) cost US $ 27, whereas rice-based production cost US $ 613 under Costa Rican conditions. Liquid culture alone would be fast but too costly: US $ 1078 ha−1. Experiments to further reduce costs in the Guata system are suggested.

Single-strain biocontrol agents often look promising when tested against single-strain pathogens. When confronted with a biodiverse field population, however, biocontrol is inconsistent. This study implies that biodiversity of the crown rot pathogen Colletotrichum musae leads to strain discrimination by antagonists which results in variable biocontrol of the disease. Broad host-range mycoparasites of fungi of the crown rot disease complex of banana (C. musae, Fusarium moniliforme and Botryodiplodia theobromae) which attacked at least two of the pathogen genera, exhibited significant differences in aggression against different strain of C. musae, the main pathogen. Antagonists acted via several different mechanisms, i.e. parasitism, antibiosis or competition, simultaneously. The relative importance of each mechanism differed with the individual mycoparasites. Strain discrimination was correlated to differential susceptibility to one or more minor mechanism(s). When as many as four antagonists were combined into one inoclum, they complemented rather than antagonised each other. Biocontrol efficiency increased with the number of antagonist strains combined. Therefore, strain mixtures should be sought to control the corwn rot disease complex of banana.