Brian R. Kerry

Potential side effects of insect-resistant transgenic plants on arthropod natural enemies

Engineering genes encoding insecticidal proteins into crop plants offers numerous benefits to agriculture. However, like many conventional insecticides, this new technology has the potential to disrupt natural biological control through both direct and indirect side effects of the plants on the fitness or behaviour of arthropod predators and parasitoids. Interactions between transgenic plants and these beneficial insects are being assessed to avoid incompatibility.

Detection and Quantification of Plectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting

ABSTRACT Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately

The Importance of the Host Plant on the Interaction Between Root-knot Nematodes Meloidogyne spp. and the Nematophagous Fungus, Verticillium chlamydosporium Goddard

300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ.

The effect of the host plant on the efficacy of Verticillium chlamydosporium as a biological control agent for root-knot nematodes was investigated in four experiments. The growth of the fungus in the rhizosphere differed significantly with different plant species, the brassicas kale and cabbage supporting the most extensive colonization. The presence of nematodes in roots increased the growth of the fungus on most plants, and this effect was associated with the emergence of egg masses on the root surface; the presence of Meloidogyne incognita did not stimulate growth of the fungus in the rhizosphere until 5 weeks after the addition of infective juveniles to soil. The susceptibility of the plant host to M. incognita attack influenced the numbers of nematode eggs parasitized by the fungus. The control of the nematode was less effective on tomato roots, which produced large galls as a result of nematode infection compared with control on potato roots where galls were smaller, despite the greater abundance o...

PCR-based molecular discrimination of Verticillium chlamydosporium isolates

The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.

Infection of plant-parasitic nematodes by nematophagous fungi – a review of the application of molecular biology to understand infection processes and to improve biological control

PCR-based assays were performed to resolve the genetic variation between 28 different isolates of Verticillium chlamydosporium using primers designed to amplify ribosomal internal transcribed spacers (ITS) and intergenic spacers (IGS). Different isolates of V. chlamydosporium were also differentiated using primers matching enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) elements. Restriction fingerprinting of PCR-amplified ITS products failed to yield intraspecific polymorphism, and different levels of discrimination between V. chlamydosporium isolates were not achieved. However restriction patterns of ITS products digested with Hae III and Hinf I were useful in differentiating between some of the closely related isolates of V. chlamydosporium, plant pathogenic Verticillium species and some common soil fungi. PCR amplification of IGS was found to be the most sensitive method which enabled the detection of 22 variants within the sample of 28 isolates of V. chlamydosporium and six different plant pathogenic Verticillium species. By using ERIC and REP-PCR fingerprinting, isolates were categorized in 20 and 13 genotypes, respectively. In general, PCR-based procedures can differentiate between closely related isolates of V. chlamydosporium within IGS genotypes. This also could be achieved by ERIC and REP-PCR, and may be considered a rapid tool for the genetic characterization and detection of different isolates of V. chlamydosporium.

Fungal parasites of cyst nematodes

Environmental concerns over conventional nematicides have led to increasing interest in the use of biological control agents to control plant-parasitic nematodes. The development of nematophagous fungi as biological control agents has revealed a need for further understanding of their infection processes. The egg-parasitic fungi, Pochonia chlamydosporia and Paecilomyces lilacinus, and the nematode trapping fungus, Arthrobotrys oligospora, have received the most attention. Through the application of biochemistry and molecular biology, aspects of their infection processes have been elucidated. This has involved the characterisation of enzymes that aid penetration of the eggshell or the nematode body wall and the identification of nematicidal toxins. This growing understanding of the biology of infection is opening new avenues in the improvement of fungi as biological control agents.

Quantification in Soil and the Rhizosphere of the Nematophagous Fungus Verticillium chlamydosporium by Competitive PCR and Comparison with Selective Plating

Abstract Cyst nematodes are major pests of a range of crops grown throughout the world. In the last decade it has been demonstrated that nematophagous fungi can effectively control some cyst-nematode pests on crops grown in monocultures. However, such natural control may be slow to establish in soil and difficult to exploit. Cropping history, summer rainfall and soil texture may affect the activity of these fungi, but their manipulation is severely restricted by a lack of detailed information on the factors that affect their growth and survival in soil. Populations of the cereal cyst nematode (CCN) may be controlled by two fungi, Nematophthora gynophila Kerry and Crump and Verticillium chlamydosporium Goddard when cereals are grown in monocultures. These fungi parasitise nematode females and eggs. The natural control of CCN has the characteristics of an “induced suppression”, in which it is essential for the host pest to be numerous in the early years of monoculture to support the multiplication of its parasites; during this time other control measures will be necessary to prevent large yield losses. Natural control caused by parasitic fungi may be difficult to measure but several techniques have been devised and in soils suppressive to CCN it has been estimated that 97% of females and eggs are parasited. Approximately 150 species of fungi have been found colonising cysts, females, and eggs of 8 species of cyst nematodes. The time of sampling and the method of isolation may affect the number and species of fungi collected. Few have been tested for their parasitic status and little is known about infection mechanisms. Some fungi have been added to soil and caused significant reductions in nematode populations but none have provided predictable control at rates of application that could be considered practical for an arable farmer. Fungi that attack eggs are facultative parasites, whereas some species are obligate parasites of females and survive in soil only as resting spores. A knowledge of the survival mechanism in soil is essential for choosing the appropriate method of applying a potential biological-control agent. The successful introduction of such an agent depends on whether a suitable niche for the organism exists or can be created and until we know much more about the factors that affect the activity of nematophagous fungi in soil, their full potentials as control agents for cyst nematodes will not be realised.

Verticillium chlamydosporium as a biological control agent for Meloidogyne incognita and M. hapla in pot and micro-plot tests

ABSTRACT A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A γ-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in γ-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.

Growth and survival of Verticillium chlamydosporium goddard, a parasite of nematodes, in soil

Verticillium chlamydosporium multiplied in peaty-sand from inoculum rates of 500, 1,000, 5,000 or 10,000 chlamydospores/g soil to a maximum number of 5.5 × 104 cfu/g soil and survived for at least 8 wk in pots planted with tomato plants inoculated with 1,000 second stage juveniles of Meloidogyne incognita. The fungus survived, but did not multiply, in loamy sand or sand. Establishment of V. chlamydosporium on the rhizoplane of tomato plants was greater in peaty sand than in loamy sand or sand. Nematode control was in general greater in peaty sand (average 59% control) than in the other two soil types (average control in loamy sand 51% and in sand 39%). In a microplot experiment on sandy loam, V. chlamydosporium controlled populations of M. hapla on tomato plants by more than 90%. The fungus multiplied and survived in soil for at least 123 days. More fungus was found in rhizosphere soil than in non-rhizosphere soil. Combining V. chlamydosporium with an aldicarb treatment equivalent to 2.8 kg a.i./ha did not affect the activity of the fungus, and gave better control (98%) than a treatment with V. chlamydosporium or aldicarb alone (90%).