Effects of disease control by fungicides on greenhouse gas emissions by U.K. arable crop production.

BACKGROUND: The U.K. government has published plans to reduce U.K. agriculture's greenhouse gas (GHG) emissions. At the same time, the goal of global food security requires an increase in arable crop yields. Foliar disease control measures such as fungicides have an important role in meeting both objectives. RESULTS: It is estimated that U.K. winter barley production is associated with GHG emissions of 2770 kg CO2 eq. ha(-1) of crop and 355 kg CO2 eq. t(-1) of grain. Foliar disease control by fungicides is associated with decreases in GHG emissions of 42-60 kg CO2 eq. t(-1) in U.K. winter barley and 29-39 kg CO2 eq. t(-1) in U.K. spring barley. The sensitivity of these results to the impact of disease control on yield and to variant GHG emissions assumptions is presented. Fungicide treatment of the major U.K. arable crops is estimated to have directly decreased U.K. GHG emissions by over 1.5 Mt CO2 eq. in 2009. CONCLUSION: Crop disease control measures such as fungicide treatment reduce the GHG emissions associated with producing a tonne of grain. As national demand for food increases, greater yields as a result of disease control also decrease the need to convert land from non-arable to arable use, which further mitigates GHG emissions.

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi.

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.

Pathogenesis, parasitism and mutualism in the trophic space of microbe-plant interactions.

Microbe-host interactions can be categorised as pathogenic, parasitic or mutualistic, but in practice few examples exactly fit these descriptions. New molecular methods are providing insights into the dynamics of microbe-host interactions, with most microbes changing their relationship with their host at different life-cycle stages or in response to changing environmental conditions. Microbes can transition between the trophic states of pathogenesis and symbiosis and/or between mutualism and parasitism. In plant-based systems, an understanding of the true ecological niche of organisms and the dynamic state of their trophic interactions with their hosts has important implications for agriculture, including crop rotation, disease control and risk management.

Production of extracellular enzymes by different isolates of Pochonia chlamydosporia.

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p<0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p<0.05), and significant differences were found between isolates (p<0.001) and the amounts of enzymes produced (p<0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.

Development of species-specific diagnostic primers for Zoophthora radicans and Pandora blunckii; two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella.

Species-specific primers for Zoophthora radicans and Pandora bluckii were developed. To achieve this, partial sequences of DNA that encode for rRNA, more specifically, the ITS region (rDNA-ITS) were obtained from different isolates and analysed. Seven Z. radicans isolates (four from P. xylostella, and three from other lepidopteran hosts) and one P. blunckii isolate (from P. xylostella) were used. These isolates were selected based on PCR-RFLP patterns obtained from 22 isolates of P. blunckii and 39 isolates of Z. radicans. All P. blunckii isolates were from the same host (P. xylostella); 20 isolates were from Mexico, one from the Philippines, and one from Germany. The Z. radicans isolates were more diverse in geographical origin (Mexico, Kenya, Japan, New Zealand, Australia, Taiwan, Philippines, Malaysia, Uruguay, France, USA, Poland, Indonesia, Switzerland, Israel, China, and Denmark) and host origin (Lepidoptera, Hemiptera, Hymentoptera, and Diptera). Using conventional PCR, each pair of species-specific primers successfully detected each species of fungus from DNA extracted from infected host larvae either single- or dual-inoculated with both fungal species. The PCR-RFLP analysis also showed that Z. radicans was genetically more diverse than P. blunckii, although only a limited number of P. blunckii isolates from one country were considered. There was no direct relationship between genetic diversity and host or geographical origin. The relationship between genetic variation within both fungal species and host specificity or ecological adaptation is discussed.

PCR to predict risk of airborne disease.

Plant, animal and human diseases spread by microscopic airborne particles have had major economic and social impacts during history. Special air-sampling devices have been used to collect such particles since the 19th century but it has often been impossible to identify them accurately. Exciting new opportunities to combine air sampling with quantitative PCR to identify and count these particles are reviewed, using crop pathogen examples. These methods can be used to predict the risk of unexpected outbreaks of airborne diseases by identifying increases in pathogen inoculum or genetic changes in pathogen populations that render control ineffective. The predictions can provide guidance to policymakers, health professionals or the agricultural industry for the development of strategies to minimise the risk of severe pandemics.

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus.

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

Development of a transformation system for the nematophagous fungus Pochonia chlamydosporia.

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

Fungal molecular diagnostics: a mini review.

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.

Approaches for monitoring the release of Pochonia chlamydosporia var. catenulata, a biocontrol agent of root-knot nematodes.

Pochonia chlamydosporia var. catenulata is a potential biocontrol agent against root-knot nematodes. Diagnosis of isolates has relied on morphological identification, and is both time-consuming and difficult. beta-tubulin primers have been developed for the identification of this fungus that were specific enough to distinguish between varieties of the fungus within the same species. Separate primers have been developed for the specific detection of P. chlamydosporia var. catenulata based on ITS sequences, which were able to detect the fungus in soil from various sites in Cuba where the biocontrol agent had been added. When the PCR diagnosis was combined with serial dilution of soil samples on selective medium, colonies were rapidly identified. The fungus was still present, albeit at low densities, in soils inoculated five years previously. The development of a baiting method allowed quick in situ screening of the isolates' ability to infect nematode eggs, and when combined with PCR diagnosis both varieties of the fungus could be detected in infected eggs. RFLP analysis of ITS sequences from P. chlamydosporia provided an extra level of discrimination between isolates.

Development of a new management strategy for the control of root-knot nematodes (Meloidogyne spp) in organic vegetable production.

The nematophagous fungus, Pochonia chlamydosporia (Goddard) Zare & Gams, has been investigated as a potential biological control agent for use in integrated pest management strategies for Meloidogyne incognita (Kof & White) Chitwood in vegetable crops. The release of the fungus as a biological control agent requires a diagnostic method to monitor its spread in the environment and to gain knowledge of its ecology. Only molecular methods are sufficiently discriminating to enable the detection of specific isolates of fungi in soil. A method to extract DNA from soil was developed to increase the efficacy of PCR-based diagnostic tests that use specific primers. A selected isolate of P chlamydosporia var catenulata was applied at densities similar to those that occur naturally in nematode-suppressive soils. The fungus significantly reduced nematode infestations in soil following a tomato crop, in a strategy that combined the use of the fungus with crop rotation. The survival of the fungus in soil was also examined in controlled conditions in which it remained in soil in densities significantly greater than its original application rate for at least 5 months. Hence, it seems that populations of this fungus may be built up in soil and have significant effects on the regulation of root-knot nematode populations.