BSPP Presidential Meeting 2002

Plant Pathology and Global Food Security


PH Gregory Prize

Biological control of late blight of potatoes

Jane Hollywood and Richard Strange

Department of Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom
Email: j.hollywood@ucl.ac.uk

Phytophthora infestans, the causal agent of late blight, is a devastating pathogen of potatoes and tomatoes world-wide. Present control measures are expensive, environmentally undesirable and increasingly ineffective. Biological control represents a realistic solution. This investigation adapts an in vivo bioassay for the selection of soils antagonistic to late blight of potatoes. Of the eight soil samples screened, four demonstrated reproducible antagonism to the development of infection of potato tubers by Phytophthora infestans as determined by the volume of rotted tissue. Microorganisms were then isolated from suppressive soils using a variety of selective and non-selective media. In total 292 bacterial samples and 20 fungal samples were recovered from the soil samples. When they were tested individually against P. infestans using the same assay as above 15 bacterial isolates suppressed tuber rotting by >85% in at least three out of four assays. These isolates were characterised to strain level as Pseudomonas spp. (3 strains), Enterobacter spp. (7 strains), Bacillus spp. (1 strain), Pantoea spp. (2 strains), Citrobacter spp. (1 strain) and Buttiauxella spp. (1 strain) by routine bacteriological tests, fatty acid profiling and partial sequencing of the gene encoding for 16S ribosomal RNA. Further analysis of the antagonists included determination of mode of action and population dynamics. Work is in progress towards developing a consortium of biological agents with different modes of action for application to potatoes since this strategy would avoid placing a single selection pressure on the pathogen population and would therefore promote longevity of the protectants.


Molecular analysis suggests sexuality in asexual fungus Fusarium culmorum

Prashant K. Mishra, Roland T.V. Fox and Alastair Culham

School of Plant Sciences, The University of Reading, RG6 6AS, UK

Asexual fungus Fusarium culmorum (W.G. Smith) Sacc is a serious pathogen that causes a variety of diseases in crop plants and is responsible for an extensive loss to agriculture. It is also known to produce trichothecene and estrogenic mycotoxins resulting in toxin contamination of crop products. The consumption of mycotoxins contaminated food and feed products pose an acute risk to human and animal health. We have analysed seventy-five isolates of Fusarium culmorum collected from different geographical locations and/or host plants using various molecular markers. The genotypic data was used to calculate the index of association, a measure of linkage disequilibrium, which suggested substantial recombination within the populations of Fusarium culmorum. Further we have shown that Fusarium culmorum possesses genes that are functional and resemble to the master sexual cycle regulators MAT1 and MAT2 of the mating type (MAT) locus of Gibberella zeae, a teleomorphic stage of Fusarium graminearum. We amplified and sequenced the alpha domain of MAT1 and high mobility group (HMG) domain of MAT2 genes from Fusarium culmorum using gene specific primers designed based on published mating type sequences of ascomycete fungi. The Fusarium culmorum MAT1 segment codes for a protein, similar in sequences to the Gibberella zeae MAT1 alpha domain protein (62% identity and 75% positives), and Fusarium culmorum MAT2 segment codes for a high mobility group protein, similar to the Gibberella zeae MAT2 HMG domain (95% identity and 100% positives). Expression profile using northern (RNA) analysis showed that both mating types genes MAT1 and MAT2 are functional in Fusarium culmorum. This is the first report concerning the identification and functional analysis of mating type genes in Fusarium culmorum.


Epidemiology and global distribution of A-group and B-group Leptosphaeria maculans on oilseed rape

Yongju Huang1, Bruce Fitt1, Avice Hall2

IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK and 2 University of Hertfordshire, Hatfield, Herts, AL10 9AB, UK

Stem canker (blackleg), caused by Leptosphaeria maculans, is a serious disease of oilseed rape worldwide [1]. The population of L. maculans can be divided into two main sub-groups, which are often termed A-group and B-group L. maculans [2]. Previous epidemiology work has mainly been on the A-group, with little information about the B-group. Previous work has mainly used conidia of L. maculans, rather than the ascospores, which play the major role in the epidemiology of the disease. In experiments, ascospores of both A-group and B-group L. maculans germinated on water agar and leaf surfaces at 5C to 20C, but B-group ascospores germinated faster than A-group ascospores. Under the same conditions, B-group ascospores produced longer, thinner germ tubes, and fewer (3.1) germ tubes per ascospore than A-group ascospores (3.8). After 24 h of incubation, the B-group hyphae grew in almost straight lines, whilst the A-group hyphae grew tortuously. Hyphae from both A-group and B-group ascospores penetrated leaves mainly through stomata, but appresorium-like structures were observed with the A-group and not the B-group. The percentage of germinating ascospores which penetrated stomata was greater for A-group than B-group L. maculans. In controlled environments, ascospores of both A-group and B-group L. maculans matured faster on infected debris at 15-20C than at 5-10C. At 5-10C, maturation of ascospores of B-group was slower than that of A-group L. maculans, but at 15-20C there was no difference between them. After burial in sand for 2, 4, 6, 8, 10 or 12 months, ascospores were produced on stem base debris but not on upper stem debris. Furthermore, only A-group L. maculans was isolated from debris after it had been buried; no B-group L. maculans was isolated. Under natural conditions, differences between seasons in time to maturation of ascospores were related to differences in epidemic development. In the UK, a large proportion of the leaf spot lesions in early autumn were caused by the A-group, but the proportion of B-group isolates obtained from stems increased during spring; A-group was predominant in the pith and cortex of the stem base, while B-group was predominant in the cortex of the upper stem. This work reported quantified important differences between A-group and B-group L. maculans, which illustrates the differences in the behaviour of the two groups in field. The distribution of A-group and B-group L. maculans differs between geographical locations. Only A-group L. maculans isolates have been found in Australia, and only B-group L. maculans have been identified in China. B-group L. maculans predominates in eastern Europe, while A-group L. maculans predominates in western Europe and North America. Since the A-group is more damaging than the B-group, strategies for preventing spread of A-group L. maculans into new areas will be discussed in this presentation.

References

[1] West J.S., Kharbanda P., Barbetti M.J. & Fitt B.D.L. 2001. Plant Path. 50:10-27.
[2] Williams R.H. & Fitt B.D.L. 1999. Plant Path. 46: 161-175.


A collection of kinases in barley powdery mildew

Gemma Priddey

Department of Plant Sciences, University of Oxford (BBSRC/CASE Studentship, in association with Aventis)

Blumeria graminis f. sp. hordei is the fungal pathogen that causes powdery mildew disease of barley. It is of significant agricultural importance, causing millions of pounds worth of crop loss every year. However, due to its obligately biotrophic lifestyle, B. graminis remains highly intractable. Therefore, novel techniques have been implemented for its study. The elucidation of signalling pathways involved in infection structure differentiation may give some insight into fungicide design. Germling morphogenesis in B. graminis is a highly regulated process, demanding the coordinate perception of external signals and their integration via complex signal transduction cascades. I have exploited the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), which invokes both germling morphogenesis and enhances PKC activity, to define a role for PKC in infection structure differentiation. This culminated in the identification of B. graminis genes involved in signal transduction processes, including two protein kinase C (PKC) genes and two mitogen-activated protein kinase (MAPK) genes. Furthermore, I have demonstrated MAPK activity in germlings and have transformed the rice blast fungus mutant, Magnaporthe grisea pmk1, with the B. graminis MAPK homologue, Mpk1, to see if it complements the lesion.


Elucidation of the interaction between the fungal pathogen Rhizoctonia solani and the cyst nematode Globodera rostochiensis in potatoes

MA Back, P Jenkinson and PPJ Haydock

Nematology and Entomology Group, Crop and Environment Research Centre, Harper Adams University College Shropshire, TF10 8NB, UK

The potato cyst nematodes (PCN) (Globodera rostochiensis and G. pallida) and the soil-borne fungus Rhizoctonia solani are both problematic and widespread pathogens of the potato crop. Synergistic interactions between these organisms have been previously reported, but until now, no study has investigated their association under field conditions, nor has any attempt been made to elucidate the mechanisms behind the interactions found. The results from two field experiments conducted during 2000 and 2001 consistently showed that the severity of stem canker and stolon canker caused by R. solani significantly increased with increasing infestations of potato roots by G. rostochiensis. Regression analysis revealed a strong and positive relationship between the number of nematode juveniles present in the roots and the incidence of stolon infections by R. solani infection of stolons by R. solani (R2=0.55). Data from the most recent field experiment (2001) has also shown a positive relationship between the incidence of root cankers and PCN. Current work is investigating the hypothesis that damage inflicted on plant roots during nematode invasion, causes an elevated release of plant compounds attractive to R. solani. Two controlled environment experiments have been initiated to determine (1) the effect of root exudates from PCN infested and non-infested plants on the growth of R. solani and (2) the effect of PCN infestations on root exudate constituents such as carbohydrates.


Molecular characterisation of virulence genes on pathogenicity islands in Pseudomonas savastanoi pv.phaseolicola

Hassan Ammouneh, Robert W. Jackson* and John.W. Mansfield

Agricultural Science Department ,Imperial College at Wye, University of London, Wye,TN25 5AH, UK.*Department of Plant Science, University of Oxford, South Parks Road, Oxford, OX1 3RB,UK.

Pseudomonas savastanoi pv. phaseolicola (Pph) causes halo blight disease in French bean (Phaseolus vulgaris L.). A region of about 30 kb on an indigenous plasmid (pAV511) from Pph race 7 strain 1449B has been found to contain a pathogenicity island (PAI). The cloned PAI on pAV520, a pLAFR3- based genomic clone, restored virulence to a plasmid-cured strain of Pph (designated RW60) which was no longer able to cause disease in cultivars of bean or soybean. Transposon mutagenesis with Tn3 gus was used to identify genes on pAV520 which had virulence function. The insertion mutagenesis strategy was co-ordinated with acquisition of the DNA sequence of the PAI. The function of genes located by transposon mutagenesis and sequencing was tested after sub-cloning of open reading frames into broad host range shuttle vectors. Several effector genes within the PAI appeared to operate synergistically in promoting virulence. One gene virPphA was effective alone. Three homologues of virPphA , named virPphApgy, virPphApsv, and avrPtoB were isolated from P.s.pv.glycinea, P.s.pv.savastanoi, and P.syringae.pv.tomato, respectively. The homologues all restored virulence to RW60 as measured by development of water-soaked lesion in bean pods and increased bacterial populations in leaves compared with RW60 alone. RW60 harbouring virPphA or virPphApsv, elicited a strong HR in soybean cv. Osumi, the presence of avrPtoB caused a weak HR, but virPphApgy, did not affect the null reaction observed in soybean with RW60 alone. In Arabidopsis, avrPtoB was the only homologue which promoted the multiplication and survival of RW60 in planta. Yeast two hybrid screens were used to identify protein targets in Arabidopsis that interact with AvrPtoB and VirPphA. Putative interactors were recovered, including a receptor like-kinase which may have an important role in plant defence.


Characterisation of the phytopathogen Pseudomonas syringae pv. tabaci

Nancy Mapuranga, CABI Bioscience, UK Centre, Egham/ Royal Holloway University of London , Egham; Julian J. Smith1; Gerry S. Saddler2; Peter M. Bramley3

1
CABI Bioscience, UK Centre, Egham. 2Scottish Agricultural Science Agency, Edinburgh, Scotland.3Royal Holloway, University of London , Egham.

Tobacco is the backbone of Zimbabwes economy and is the countrys major single foreign currency earner accounting for about 50% of Zimbabwes foreign currency earnings. The country rates third behind the United States of America and Brazil in total exports of all types tobacco. Tobacco production employs a third of all the people working in the agricultural sector, making it the single largest employer in this sector with about 700 000 people dependent on tobacco for their livelihood. The crop is however severely affected by foliar bacterial diseases like wildfire and angular leaf spot, caused by two forms of the same pathogen, Pseudomonas syringae pv. tabaci. These diseases cause economic losses in yield and quality every year. A poor understanding of the pathogen compromises the efficiency and appropriateness of management strategies in control. Phytopathogenic bacteria of the Pseudomonas syringae group are traditionally divided into pathovars on the basis of host range and type of symptoms; race structures within pathovars are also recognised based on the reaction of differential host cultivars. These approaches are both time consuming and unreliable hence more rapid identification methods are required for use in disease diagnosis. Accordingly, phenotypic and genotypic based methods were evaluated to differentiate between pathovars of P. syringae and closely related species. A total of 114 field isolates including reference isolates of fluorescent Pseudomonas species belonging to P. syringae pathovars atrofaciens, coronafaciens, garcae, glycinea, helianthi, lachrymans, maculicola, phaseolicola, pisi, striafaciens, syringae, tabaci, tomato and related Pseudomonas cichorii were characterised. Phenotypic methods, which included the LOPAT determinative scheme, fatty acid analysis by gas chromatography, Biolog and API confirmed species designation but failed to resolve between pathovars. It was however observed that the P. s. pv. phaseolicola and P. s. pv. glycinae isolates formed a distinct cluster whereas P. s. pv. tabaci isolates were more diverse across three broad clusters. Genotypic characterisation by "whole cell" rep-PCR using the primers BOX and ERIC gave distinct fingerprint profiles. Cluster analysis on the combined data sets generated from the two primers successfully separated most isolates into pathovar distinct groups. A parallel assessment using AFLP is underway, providing complementary data sets. These data sets will be compared with race designation determined by differential cultivar testing to determine whether a race structure is evident within the DNA profiles of rep-PCR and/or AFLP.


PCR based quantification of Verticillium dahliae in soil

Vinodh Krishnamurthy

Horticulture Research International, Wellesbourne and the University of Aberdeen.

Verticillium wilt in strawberries is an economically important disease caused by soil-borne fungus Verticillium dahliae. A PCR-based soil test for the quantification of V. dahliae is being developed to overcome problems with the wet plating method. This test is intended for use in assessing the risk of verticillium wilt in potential planting sites for strawberry. A competitive PCR assay was developed for V. dahliae DNA from mycelium. The test uses V. dahliae specific primers developed from a clone from a random genomic library. A heterogeneous competitor DNA fragment, for use as an internal standard was developed from N. tabacum DNA amplified under low stringency annealing conditions. Comparison of band intensity following co-amplification of V. dahliae DNA from mycelium with 2fg of competitor DNA provides accurate quantification in the range of 2-200pg and a detection limit of approximately 60 genomes. The sensitivity and the dynamic range of starting target molecule determined using this assay is being compared with an automated PCR using SYBR Green I. Currently the Ultraclean soil DNA isolation kit followed by a Qiagen PCR purification kit is being used as a simple and rapid method for obtaining PCR-quality DNA from microsclerotia in soils. We are trying to estimate an approximate number of genomes in microsclerotia in order to relate PCR-based results with infectivity. Fresh microsclerotia produced in vitro in four different size ranges are currently being used but we will later study microsclerotia from soils.


An optimised inoculation method to screen cacao (Theobroma cacao L.) for resistance to Witches Broom Disease caused by Crinipellis perniciosa

S. Surujdeo-Maharaja*, P. Umaharana and D.R. Butlerb.

a
Department of Life Sciences; bCocoa Research Unit; Faculty of Agriculture and Natural Sciences, University of the West Indies, St. Augustine.

The effects of host-(leaf number, clone/seedling), pathogen-(spore-concentration) and environmental (incubation time) factors on inoculation success to Witches Broom Disease were investigated in greenhouse experiments using susceptible cacao genotypes. Three methods of inoculation (agar-drip, water-drip and spray) were also tested for inoculation success against the pathogen. The optimised inoculation method was tested for its repeatability as well as its ability to discriminate between various levels of resistance to C. perniciosa in cacao. The optimised method (350,000 viable basidiospores/ml; 60h incubation; agar-drip technique) was able to produce 100% infection on both clonal and seedling plants of a susceptible genotype, on a repeatable basis. Leaf number did not significantly affect percentage symptomatic plants or broom characteristics. This method effectively discriminated between the various levels of resistance in 14 cacao genotypes and is recommended as a screening method to identify levels of resistance in germplasm collections. Symptom severity has been shown to be a better measure of resistance than infection success.

*E-mail: kitataka@hotmail.com


Characterization of a collection of flax-root-colonising fungi and their implication in root rots.

E. Cariou1, S. Foucher2, G. Raynal3, and Y. Brygoo2

Institut Technique du Lin, Boite 52, 15, rue du Louvre, 75001 Paris, ecariou@lin-itl.com 1 Laboratoire de Pathologie, Mthodologie et Mthodes de Dtection, INRA, route de Saint Cyr, 78 Versailles, 2 and Laboratoire de Pathologie Vgtale, INAPG-INRA, 78 Thiverval Grignon, 3 France.

The origin of flax (linum usitatissimum) scorch disease or root rot remained uncertain and even controversial since the beginning of the 20th century. In order to identify pathogens, a collection of fungal isolates from field-infested flax plants was established. These fungi were initially grouped according to their colony morphologies. Representative isolates were selected for pathogenicity tests. The fungi were assessed for their abilities to induce root symptoms on flax young plants to determine which group of fungi was responsible for flax scorch disease, in regards to plant symptoms from infected-fields.

In order to identify flax root colonising pathogen fungi, RFLP analysis of nuclear ribosomal DNA was performed with four enzymes HhaI, HinfI, NdeII and HaeIII. RFLP of unknown fungi were compared with RFLP from species isolates belonging to several collections meanly from Laboratoire de Pathologie, Mthodologie et Dtection de Versailles. A total of 81 isolates were clearly identified. The sequence of ITS region in 6 unknown isolates with no common RFLP results referred in the French collection of Versailles were resolved.