Optimised and systematic suitable climate modelling confirms future longitudinal-trends for growing oil palm in Africa.

Palms are iconic plants. Oil palms are very important economically and originate in Africa where they can act as a model for palms in general. The effect of future climate on the growth of oil palm will be very detrimental. Latitudinal migration of tropical crops to climate refuges may be impossible, and longitudinal migration has only been confirmed for oil palm, of all the tropical crops. The previous method to determine the longitudinal trend for oil palm used the longitudes of various countries in Africa and plotted these against the percentage suitable climate for growing oil palms in each country. An increasing longitudinal trend was observed from west to east. However, the longitudes of the countries were randomly distributed which may have introduced bias and the procedure was time consuming. The present report presents an optimised and systematic procedure that divided the regions, as presented on a map derived from a CLIMEX model, into ten equal sectors and the percentage suitable climates for growing oil palm were determined for each sector. This approach was quicker, systematic and straight forward and will be useful for management of oil palm plantations under climate change. The method confirmed and validated the trends reported in the original method although the suitability values were often lower and there was less spread of values around the trend. The values for the CSIRO MK3.0 and MIROC H models demonstrated considerable similarities to each other, contributing to validation of the method. The procedure of dividing maps equally into sectors derived from models, could be used for other crops, regions, or systems more generally, where the alternative may be a more superficial visual examination of the maps. Methods are required to mitigate the effects of climate change and stakeholders need to contribute more actively to the current climate debate with tangible actions.

Failed PCR of Ganoderma type specimens affects nomenclature.

The nomenclature of Ganoderma used as a Chinese medicine is debated. A group of researchers could not amplify the DNA of type specimens and concluded the DNA was degraded irreparably. New topotypes were used as the type specimens which was premature. The use of internal amplification controls is recommended to determine if other factors were involved as alternative explanations.

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.

Biofilms from a Brazilian water distribution system include filamentous fungi.

Filamentous fungi in drinking water can block water pipes, can cause organoleptic biodeterioration, and are a source of pathogens. There are increasing reports of the involvement of the organisms in biofilms. This present study describes a sampling device that can be inserted directly into pipes within water distribution systems, allowing biofilm formation in situ. Calcofluor White M2R staining and fluorescent in situ hybridization with morphological analyses using epifluorescent microscopy were used to analyse biofilms for filamentous fungi, permitting direct observation of the fungi. DAPI (4',6-diamidino-2-phenylindole) was applied to detect bacteria. Filamentous fungi were detected in biofilms after 6 months on coupons exposed to raw water, decanted water and at the entrance of the water distribution system. Algae, yeast, and bacteria were also observed. The role of filamentous fungi requires further investigations.

Interaction with Penicillium expansum enhances Botrytis cinerea growth in grape juice medium and prevents patulin accumulation in vitro.

Interactions between fungi occur when they grow on the same host plant. This is the case of Botrytis cinerea and Penicillium expansum on grape. P. expansum is also responsible for production of the mycotoxin patulin. In this study, the influence of the interaction between both fungi on fungal growth parameters was studied as well as the effect on the accumulation of patulin by P. expansum. For that purpose, spores of B. cinerea and P. expansum were inoculated together (mixed inoculum), and the parameters growth rate, time for growth and patulin accumulation were assessed. The presence of P. expansum conidia shortened the time for growth of mixed inoculum colonies which, at the end of incubation, were B. cinerea-like. Although some P. expansum growth was observed in mixed inoculum colonies, very low levels of patulin were observed. In assays carried out in patulin-spiked medium, B. cinerea was capable to metabolize the mycotoxin. The capabilities of B. cinerea to shorten time for growth and prevent patulin accumulation are competing abilities that facilitate grape colonization.

Effects of the origins of Botrytis cinerea on earthy aromas from grape broth media further inoculated with Penicillium expansum.

Earthy "off" aromas from wine and grape juice are highly detrimental to the production of quality grape products. These volatile compounds are produced on grapes by Botrytis cinerea, Penicillium expansum and/or a combination of P. expansum and B. cinerea strains. B. cinerea strains were isolated from different (a) vineyards in Spain and Portugal, (b) grape varieties (c) bunches (i.e., sound and botrytized) and (d) positions in the botrytized bunch (i.e., interior or exterior). A novel Headspace-Phase Microextraction (SPME) followed by Gas Chromatrography/Mass Spectrometry (GC-MS) dedicated to analyze geosmin, methylisoborneol (MIB), 1-octen-3-ol, fenchone and fenchol in grape broth medium was used. Approximately 50% of the B. cinerea strains induced detectable geosmin. One strain accumulated significant amounts of anisoles, demonstrating that this contamination might already occur in the vineyard. Strains from the interior of Cainho grape bunches induced more geosmin and hence it may be possible to reduce this volatile in wine by avoiding using these grapes in case of B. cinerea attack.

Microextraction and Gas Chromatography/Mass Spectrometry for improved analysis of geosmin and other fungal "off" volatiles in grape juice.

Geosmin is a volatile fungal metabolite with an earthy aroma produced in grape products from rotten grapes. The accumulation of geosmin in grapes is caused by the interaction of Botrytis cinerea and Penicillium expansum. Solid Phase Microextraction (SPME) has great utility for collecting volatile compounds in wine. However, contamination with earthy odours may have occurred previously in the must and novel methods are required for this commodity. In the present report, several parameters of the SPME were evaluated to optimize geosmin extraction. The method permitted quantification of geosmin and other fungal volatiles by Gas Chromatography-Mass Spectrometer (GC-MS) at very low concentrations. Limits of detection and quantification (L(D) and L(Q)) for geosmin were 4.7 ng L(-1) and 15.6 ng L(-1) respectively. The RSD was 4.1% and the recovery rates ranged from 115% to 134%. Uniquely, haloanisoles were analyzed by using only one internal standard (2,3,6-trichloroanisole) thus avoiding the synthesis of deuterated anisole analogues that are used as internal standard in other methods. The method was used for the analysis of grape juice samples inoculated with B. cinerea and P. expansum. Geosmin and methylisoborneol were the compounds that appeared to contribute most to earthy odours, although other fungal compounds which are claimed to cause earthy or mouldy off-odours were detected (e.g. 1-octen-3-ol and fenchol).

Survey of aflatoxins in chillies from Pakistan produced in rural, semi-rural and urban environments.

Chilli peppers from Pakistan are consumed locally and also exported. Their quality is compromised by aflatoxins (AF) contamination. AF in chillies from rural, semi-rural and urban areas of the Punjab region of Pakistan were determined. Twenty-three (52.3%), 22 (50%) and 29 (65.9%) samples from rural, semi-rural and urban areas respectively, contained levels of aflatoxins which exceeded the European Union limits of >5 µg kg(-1) for AFB1 and >10 µg kg(-1) for total AF that apply to spices. Mean values for AFB1 in ground samples were 23.8, 14.8 and 14.0 µg kg(-1) for rural, semi-rural and urban areas, respectively. Mean total AF in ground samples were 27.7, 17.7 and 16.2 µg kg(-1) from equivalent locations. Eleven (50%), 12 (54.5%) and 14 (63.6%) whole samples from rural, semi-rural and urban areas, respectively, contained total levels of AF that exceeded European Union limits. The data indicate that individual localities have particular problems. In conclusion, the concentrations were often greater than the statutory limits set by the European Union.

Filamentous fungal characterizations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV-absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI-TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI-TOF ICMS to identify fungi are required.

Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi.

Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus, as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers.

Internal amplification controls have not been employed in fungal PCR hence potential false negative results.

Polymerase chain reaction (PCR) is subject to false negative results. Samples of fungi with the genes of interest (e.g. a disease or mycotoxin) may be categorized as negative and safe as a consequence. Fungi are eukaryotic organisms that are involved in many fields of human activity such as antibiotic, toxin and food production. Certain taxa are implicated in human, animal and plant diseases. However, fungi are difficult to identify and PCR techniques have been proposed increasingly for this purpose. Internal amplification controls (IACs) will ameliorate the situation and need to become mandatory. These are nucleic acids that posses a sequence which will provide a PCR product (i) using the same primers employed for the target gene, and (ii) that will not coincide on the gel with the product of the target gene. Only one group of workers employed an IAC, to respond to potential inhibition, which was reported in 1995 from this present assessment of numerous reports. Inhibitors in cultures need to be minimized, and secondary metabolites are an obvious source. The fields reviewed herein include medical mycology, mycotoxicology, environmental mycology and plant mycology. The conclusion is that previous reports are compromised because IACs have not been employed in fungal PCR; future research must include this control at an early stage.

Laccase activity from the fungus Trametes hirsuta using an air-lift bioreactor.

AIM: To produce high laccase activities from the white-rot fungus Trametes hirsuta in an in-house air-lift bioreactor (ALB). METHODS AND RESULTS: Trametes hirsuta was grown in a 6-l ALB. A fed-batch strategy with glycerol as an addition resulted in maximum laccase activity of 19,400 U l(-1), which was the highest reported from the fungus. CONCLUSION: The ALB configuration with additional glycerol resulted in high laccase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on how to produce high concentrations of laccase.

Mycotoxin production from fungi isolated from grapes.

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.

Mycoflora and mycotoxins in Brazilian black pepper, white pepper and Brazil nuts.

A wide range of field and storage fungi were isolated from black pepper, white pepper and Brazil nut kernels from Amazonia. A total of 42 species were isolated from both peppers. Aspergillus flavus and A. niger were isolated more frequently from black than from white pepper. Other potential mycotoxigenic species isolated included: A. ochraceus, A. tamarii, A. versicolor, Emericella nidulans and Chaetomium globosum, Penicillium brevicompactum, P. citrinum, P. islandicum and P. glabrum. Species isolated from pepper for the first time were Acrogenospora sphaerocephala, Cylindrocarpon lichenicola, Lacellinopsis sacchari, Microascus cinereus, Petriella setifera and Sporormiella minima. Seventeen species were isolated from Brazil nut kernels. A. flavus was the dominant species followed by A. niger. P. citrinum and P. glabrum were the only penicillia isolated. Species isolated for the first time included Acremonium curvulum, Cunninghamella elegans, Exophiala sp., Fusarium oxysporum, Pseudoallescheria boydii, Rhizopus oryzae, Scopulariopsis sp., Thielavia terricola and Trichoderma citrinoviride. Considerably more metabolites were detected from black than white pepper in qualitative analyses. Chaetocin, penitrem A, and xanthocillin were identified only from black pepper, and tenuazonic acid was identified from both black and white pepper. Aflatoxin G2, chaetoglobosin C, and spinulosin were identified from poor quality brazil nuts. Aflatoxin B1 and B2 were also only detected in poor quality brazil nuts at concentrations of 27.1 micrograms kg-1 and 2.1 micrograms kg-1 respectively (total 29.2 micrograms kg-1).

Mycoflora and mycotoxins of Brazilian cashew kernels.

Kernel samples of common and dwarf Brazilian cashew nuts were highly contaminated with field and storage fungi in comparison to healthy ones. In general, dwarf cashews were more contaminated than common. A total of 37 fungal species were identified. Aspergillus niger was the dominant species with more colonies being isolated from dwarf kernels. A. flavus was the next most frequently isolated species. Penicillium brevicompactum, and P. glabrum were the most frequently isolated penicillia, with higher contamination recorded from dwarf kernels. Chaetomium globosum was recorded at a high level. Nine species were recorded from cashew kernels for the first time. Multimycotoxin analysis by tlc and hplc were positive for mycotoxins and other secondary metabolites particularly from the infected samples. Hplc was only carried out on dwarf cashews. Aflatoxins were not detected by quantitative high performance thin layer chromatography.

Methods for the determination of filamentous fungi in treated and untreated waters.

Results of two recent projects in the UK and USA have shown that many species of filamentous fungi are present in both untreated and treated waters. Samples have been taken from surface waters and from a range of points within distribution networks. Isolation programmes employing a range of methods and media, combined with expert taxonomic study have yielded extensive lists of waterborne fungi identified, where possible, to species level: 141 different taxa from the UK and 140 from the USA. In the UK study an average of 32 different fungal taxa were isolated from each surface water and compared to nine from each treated tap water. Certain fungi appear more frequently than others, in particular species of Aspergillus, Cladosporium, Epicoccum, Penicillium and Trichoderma appear to be very common but species from many other genera are also present. The significance of fungi in water systems is poorly understood, many of the species isolated from water are known to be capable of producing toxic secondary metabolites, are involved in food spoilage, or are plant pathogens; some are even opportunistic human pathogens. Direct observation of isolation filters has shown that fungi are present equally as spores and as hyphae. Studies have shown that fungi are capable of growth in both treated and untreated water. While traditional methods of measurement such as dry weight are inadequate for determining growth in water, analysis of ergosterol-a universal component of cell membranes amongst filamentous fungi-has provided a very sensitive means of measuring fungal growth in water to levels of < 1 µg(-1) and may prove to be a suitable method for detecting general fungal levels in water samples.

Effect of 2-deoxy-D-glucose on mycotoxins from apples inoculated with Penicillium expansum.

Patulin concentration was not significantly different in Bramley and Cox's apples inoculated with Penicillium expansum, and treated with the biocontrol enhancer 2-deoxy-D-glucose (DOG) when compared to no DOG treatment, despite average numerical increases. Some additional small HPLC peaks were detected from some extracts, one of which corresponded to citrinin.

Natural occurrence of aflatoxins and Aspergillus flavus (Link) in water.

Aflatoxins were detected and Aspergillus flavus was identified from water from a cold water storage tank. This is the first published report of such data from water.

Effect of fungistatic agent 2-deoxy-D-glucose on mycotoxins from Penicillium expansum.

Mycotoxin production from Penicillium expansum was effected by the 'biocontrol enhancer' 2-deoxy-D-glucose (DOG). Citrinin and gentisyl alcohol were produced in higher amounts, whereas patulin was inhibited, which represents a differential effect on the polyketide metabolic pathway. These effects have implications for the use of DOG in biocontrol.

Neutral, alkaline and difference ultraviolet spectra of secondary metabolites from Penicillium and other fungi, and comparisons to published maxima from gradient high-performance liquid chromatography with diode-array detection.

The ultraviolet spectra of 6 predominantly secondary metabolites from filamentous fungi which, inter alia, are useful in the identification of the compounds after chromatography, were obtained in neutral (methanol) and alkaline solvents. Difference spectra were obtained by subtracting the neutral from the alkaline spectrum for each metabolite, using the spectrophotometer software. The data and method are of use in differentiating metabolites with similar chromophores. A database of the maxima was stored on a microcomputer for flexible storage, retrieval and updating of information. These data are compared to those published previously, obtained by diode-array detection using gradient high-performance liquid chromatography, which indicated that changes in solvent concentrations of the gradient affect the spectra of some metabolites. This could cause misidentification of chemosyndromes and metabolites which have been claimed to be of use in fungal chemotaxonomy.