Simon D. Atkins

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.

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.

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 Kendalls 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.

Measuring abundance, diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia

Abstract Abundance, genetic diversity and parasitic ability in the facultative nematode parasite Pochonia chlamydosporia var. chlamydosporia were compared in soils from two sites in Portugal under long-term tomato cultivation where root-knot nematodes (Meloidogyne sp.) were present. Fungal abundance assessed by selective agar or real-time quantitative PCR with specific primers was similar in both soils. PCR fingerprinting of isolates with ERIC primers indicated that the dominant P. c. var. chlamydosporia biotypes (profiles A and B) in both soils were very closely related, although a second biotype (profile C) was detected in one soil. When tomato plants infected with M. incognita were grown in the two soils, only profiles A and B were recovered from eggs. Primers based on polymorphisms in vcp1 demonstrated that isolates with profiles A and B were likely to prefer root-knot nematodes, whereas profile C preferred cyst nematodes. In the soil containing profiles A, B and C, egg parasitism by P. chlamydosporia was estimated at 1% using water agar plates with antibiotics but fewer than 0.2% of M. incognita eggs were shown to be infected with P. c. var. chlamydosporia when using species-specific β-tubulin-PCR primers. In contrast, the soil containing only profile B showed 22% egg parasitism on water agar plates and more than 2.5% of eggs were confirmed as P. c. var. chlamydosporia by species-specific β-tubulin-PCR primers. The results, which reveal limited diversity within the fungus at the two sites, are discussed in relation to biological control of plant-parasitic nematodes.

A molecular diagnostic method for detecting Nacobbus in soil and in potato tubers

Species of the genus Nacobbus have the potential to reduce yields of major food crops such as potato, sugar beet and tomato in many parts of the world, thus warranting a quarantine effort to avoid their introduction. Molecular studies offer a new method for routine quarantine diagnostics for this nematode that will be faster and more sensitive than previous methods. A primer set was designed from Nacobbus ITS sequences and their specificity confirmed. DNA was extracted from nematodes, soil and potato tubers for use in PCR. Optimised PCR conditions were established and the PCR products were separated on 2% agarose gels, showing that specific ITS primers for the detection of Nacobbus generated a single PCR product, although band size varied slightly between species and soil isolates. The product was generated from DNA extracted from all the Nacobbus samples but not from other nematodes tested (Pratylenchus, Radopholus, Meloidogyne, Globodera, Heterodera). No bands were generated from the uninfested control soil and control tuber DNA samples, thus demonstrating the specificity of the primers. For the first time, Nacobbus was detected in soil and tuber samples using molecular approaches. These results have important applications not only in analysing advisory samples but also in the screening of material for quarantine purposes.

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.

Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres

Aims:  To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co‐infection of plants and nematodes.

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.

Genetic basis of control of Rhynchosporium secalis infection and symptom expression in barley

The genetic basis of several different components of resistance to Rhynchosporium secalis in barley was investigated in a mapping population derived from a cross between winter and spring barley types. Both the severity of visual disease symptoms and amount of R. secalis DNA in leaf tissues were assessed in field trials in Scotland in the 2007/2008 and 2008/2009 growing seasons. Relative expression of symptoms was defined as the residual values from a linear regression of amount of R. secalis DNA against visual plot disease score at GS 50. Amount of R. secalis DNA and visual disease score were highly correlated traits and identified nearly identical QTL. The genetic control of relative expression of symptoms was less clear. However, a QTL on chromosome 7H was identified as having a significant effect on the expression of visual disease symptoms relative to overall amount of R. secalis colonisation.

Effects of disease control by fungicides on greenhouse gas emissions by UK arable crop production

BACKGROUND The U.K. government has published plans to reduce U.K. agricultures 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.