BSPP News Spring 2002 - Online Edition

The Newsletter of the British Society for Plant Pathology
Number 41, Spring 2002 

8th International Verticillium Symposium  Cordoba, Spain : 5 - 8 November 2001

The 8th International Verticillium Symposium was organised by Prof Rafael Jiminez Diaz and his excellent and extensive local team. Nearly 100 participants from 20 countries gathered in that beautiful and historic city (more on that later) to discuss Verticillium. Few other plant pathogens exhibit the extraordinary plasticity of V. alboatrum and V. dahliae to infect a multitude of economically important tropical and temperate crops. New hosts continue to emerge and they remain one of the most difficult groups of pathogens to control.

Three of us from the UK received partial funding from BSPP and in this combined report we select a few items from our respective areas of interest on molecular methods (Dimitra Gkilpathi, IACR Rothamsted), host-pathogen interactions, culture and food (!) (Richard Cooper (RMC), University of Bath) and control and advisory matters and rioja (Tom Locke (TL), ADAS, UK). 

Los Amigos! Over 25 years on from Imperial College, still working with Verticillium and 
not a sign of wilting. Eris Tjamos (Athens) and Richard Cooper (Bath).

Origin of V. longisporum. R Bhat et al. (Salinas, USA) debated the issue of whether all crucifer isolates should be included in the new sp V. longisporum. Previously this distinction from V. dahliae and V. albo-atrum has been based on host specificity, longer conidia and higher DNA content. However, character- isation of cruciferous isolates suggested these isolates are a sub-group of V. dahliae. Alexander Collins et al (HRI, UK) used sequencing by PCR amplification or SSCP of ITS regions of rRNA from 30 crucifer isolates to show that at least four different hybrisation events had occurred. Japanese and European crucifer isolates appeared to be clonally derived from a single hybridisation event and occurrence in different continents suggests transportation in or within seed. USA and German isolates have arisen from different hybridisation events. Dez Barbara (HRI) presented a poster using microsatellite markers. Starting from V. longisporum, they expected markers to show different specificities and two products representing polymorph-isms between the supposed parents, but these were not evident. Aposter by Clewes et al. showed clearly though that V. albo-atrum and V.dahliae are vegetatively compatible. This could allow heterokaryons to form, opening the way to nuclear fusion and true interspecific hybrids. In one pairing microsclerotia produced at the mycelial interface were similar to those formed by crucifer isolates. (TL points out that V. longisporum still causes problems in Sweden in oilseed rape so we need to be diligent in the UK).

A lively discussion followed (after a typical fine rioja-laced lunch) about the parasexual cycle and mechanisms by which new strains arise. It was especially enhanced by the presence of Alex Hastie, who, although long retired, remembers a thing or two about Verticillium genetics.

Molecular methods and applications. Workers using isolates from different hosts described characterisation by diverse molecular methods, which complement traditional pathogenicity tests and VCG groups. Hopefully, in the future, robust, comparable techniques will be used by all working with V. dahliae populations. A useful application was described by J. Marcado-Blanco (Cordoba); RAPD analysis of defoliating and non-defoliating V. dahliae isolates from olives allowed design of specific primers to distinguish them and to develop nested PCR for early detection of disease and of pathogen-free material for planting new orchards.  Dimitra Gkilpathi showed that linseed isolates were not host-adapted; RFLP analysis of rDNA showed all linseed isolates were in the same group.

Host-pathogen interactions. At the last Symposium, RMC expressed concern and surprise at the lack of progress in the '90s in understanding interactions between vascular pathogenic fungi and their hosts. They are amenable to molecular analysis and are easily grown, even in simulated xylem fluids.  K. Dobinson (London, Ontario) took up the challenge, including 6 months in Bath in 2000. In Cordoba she described a genomic approach to analysing pathogenicity and microsclerotial formation. Over 1000 ESTs (expression tagged sequences) are revealing putative genes including wall degrading enzymes, toxins, hydro-phobins, ACC synthases, with over 40% ESTs having weak or no similarity to known sequences; some of these genes are bound to hold the key!  S. Kang (Penn State, USA) described the rewards of working with Arabidopsis to study the neglected root diseases ("model" diseases are almost invariably foliar) including V. dahliae and Fusarium oxysporum. His group is using Agrobacterium-mediated transformation of V. dahliae for random insertional mutagenesis and gene knockouts.

As well as chairing a session RMC gave a paper on production of elemental sulphur by resistant genotypes from different taxa in response to Verticillium and other vascular pathogens. Sulphur is of course highly fungitoxic and shows a pattern of accumulation typical of organic phytoalexins i.e. rapid and high in resistant, low and slow in susceptible interactions. Vascular parasitic isolates of Verticillium were not more tolerant than non-vascular isolates.Its production may be specific to xylem, as it was not detected in diseases involving other tissues. In the paper quantification of S was detailed as well as accumulation in structures that Verticillium would encounter in the xylem. Biogenesis of S is being studied by molecular analysis of gene expression. This is the first example of an element performing directly in defence and of a phytoalexin in different taxa. 

After the meeting RMC had another shot of disease mechanisms by visiting the fungal genetics group, including Antonio di Pietro and Isabel Roncero at the University of Cordoba. They are doing excellent work on F. oxysporum f.sp lycopersici by targetted and random gene knockout. I gave a seminar on microbial depolymerases in pathogenicity and unofficially opened their new lab (by cutting a parafilm tape); which means I was allowed to stay on and help them celebrate.

Jane Robb (Guelph, Canada) clearly demonstrated the phenomenon of "tolerance" whereby PCR revealed an isolate of V. dahliae pathogenic to eggplant, extensively colonised tomato but with minimal symptoms. The question still remains after so long, what are the different mechanisms in these superficially similar interactions? Are fungal virulence factors not triggered or degraded or tolerated?

There was little else on the nature of defence, but the rigours of screening for it were described by J. Hiemstra (Wageningen, Netherlands). 2,000  Norway maples (Acer platanoides) as seed from nine regions was eventually reduced to 35 promising lines following stem inoculation. Resistant clonal rootstocks may solve the problem, which was the same conclusion from the work of M Cirulli (Bari, Italy) for V. dahliae of peach. Hiemstra also coordinates an EU-funded project of 10 groups working on Verticillium wilts of trees. 

Spread and effects. Verticillium continues to spread its geographic and host ranges. Following problems in the USA on cauliflower in 1990 and horseradish in 1994, the first report on lettuce in 1995 was confirmed by K. Subbarao as a real threat in California. The disease can also be seed transmitted. TL comments that the soil inoculum levels thought to trigger infection are higher than found in the UK. 

In a poster, S. Paternotte described V. albo-atrum as a serious disease in tomatoes grown in rockwool in the Netherlands, although most vars contain the Ve gene. The symptoms could be reduced by trimming the trusses (but am I missing the point here of tomato growing? RMC). Ten similar cases have been reported this year in the UK, including by some major growers.

Problems local to Cordoba centred on cotton and olive. J. Bejarano-Alcazar et al. reported Verticillium wilt on ca. 88% of cotton fields in the Gualdalquivir Valley of Andalucia (Cordoba is sited on this river). A highly virulent defoliating (D) pathotype has spread 200 km in 6 years from the lower to central valley. Orderly stands of olives dominate the local landscape. Of the 8.2 million ha in the Mediterranean, Spain has 2.25 million ha. Again a D pathotype is increasing which can be lethal (J. Mercado-Blanco et al.) and may result from establishment of new irrigated olive orchards in Verticillium-infested soil. A modelling study by J. Navas-Cortes et al. suggested spread into olive from inoculum originating from a nearby affected cotton crop. V. Rodriguez-Morcillo et al. described the wide host range of local isolates, which underlines the limitations of crop rotation against V. dahliae. Tom Locke's poster described the potential effect of V. dahliae in England and Wales. Based on estimates of yield losses in different cultivars, levels of V. dahliae in farm soils, hectarage of each cultivar grown and the average cultivar yield in the absence of disease, ca. 740,000 tonne p.a., value £60M might be lost.

Control: bugs, manure, volatiles and herbs. In the absence of resistant genotypes, disease control against such an intractable genus continues to exercise many groups and some resort to extreme practices, from cutting off its oxygen supply to choking it. At the milder end, various biocontrol agents have been identified , some resulting in encouraging decreases in disease: Talaromyces (syn Penicillium dangeardii) (D. Fravel & Bao, Beltsville, USA), Serratia plymuthica (a commercial product is emerging) and Strentrophomonas maltophila  (S. Kurze et al. Rostock, Germany), Paenibacillus  (Antonopoulos et al. Athens, Greece), fluorescent Pseudomonas spp (Mercado-Blanco et al, Cordoba). One group set a thief to catch a thief: Varela et al. (Coruna, Spain) found that some protection was given to pepper against V. dahliae by preinoculation with F. oxysporum f.sp lycopersici. Presumably tomatoes would not be part of the rotation!

Amendments to soil may sound like old technology but some are clearly effective. In Israel, Verticillium in potatoes has been controlled by green manure (L. Tsor, Gilat) or combined NH4 and limed sludge (A. Gips et al., Volcani). In the Netherlands deinfestation of tree nurseries and strawberry fields was attempted with green manure, mulching and airtight plastic to create anaerobic conditions; V.dahliae inoculum was reduced by 75-90% (J Gould et al. and J. Lamers et al.).  Addition of fresh broccoli residues is now accepted as an effective way of reducing V. dahliae propagules in soil (broccoli is resistant to Verticillium). The possible active components were investigated as S-containing volatiles from 15 Brassica spp., incorporated into soil by GC by D Harris & R Murray (HRI, UK). The effect did not appear to be due to the action of isothio-cyanates derived from glucosino-lates, but rather S-containing volatiles such as methanethiol.
The enormous problem of acceptable disposal in Canada of considerable volumes of liquid swine manure (well what would you call it?) might be combined with its ability in some soils to control V. dahliae. G Lazarovits et al. (Ontario, Canada) described that the active components appear to be volatile fatty acids under acidic conditions. The Spanish way as described by C. Mwanza & M. Blanco-Lopez (Cordoba) seems much sweeter, with various organic supplements, which included pine needles, thyme and eucalyptus; the treatments vary greatly in reducing viability of micro-sclerotia but the fields smell wonderful.
Wilting families. Family bonds are strong in this part of the world and it was evident in the paper presented by E Tjamos and son and that Rafael Jiminez's daughter is completing a PhD on Fusarium wilt of chickpea. I (RMC) can just imagine my daughter and I surviving such an experience!

Cordoba, birds, art, oil, food and wine. Some of us arrived early enough to enjoy superb weather, but most had to deal with two days of rain before the skies cleared again. It wasn't all work, and the organisers ensured that we overdosed on food, wine and culture. Cordoba was declared a World Heritage town by UNESCO in 1995. The old town is well preserved and labyrinthine; the conference was on the edge of the maze of streets that is the Juderia, complete with ancient synagogue. The city reflects Islamic Spain's peak with independence from Baghdad declared in 929. Then Cordoba was the biggest city in Western Europe with a population between 100-500,000; now it has about 350,000 inhabitants. There is a splendid, much restored Roman bridge spanning the river; from here one can watch each evening a remarkable display of vast numbers of egrets and starlings, which come to roost on the islands by the Islamic water mills. Throughout the old city, concealed behind wooden doors or wrought iron gates, are many examples of a fine Cordoban tradition. Long have their patios provided shade from the fierce summer heat. Many are open in May for a competition but any time one can view twelve great examples in the Palacio de Viana. We were given a tour there on Wednesday evening. Of the famous Cordo-ban scholars and artists, RMC's favourite is the Jewish philosopher Maimonides (1135-1204) whose work has the splendidly Python-esque title of " Guide for the Perplexed"; anyone working with Verti-cillium might seek solace in this. More recently the artist Julio Romero de Torres (1880-1930) has a museum dedicated to him where are housed his dark, sensual portraits of Cordoban women. "Naran-jas y limones" puts Nell Gwynne holding fruits strategically to shame. Conference lunches were in the beautiful Palacio de Congresos y Exposiciones within the Bishops' Palace. This fine building is across from the world famous Mezquita Catedral (mosque-cathedral). There was a guided tour of this truly remarkable, predominantly Islamic structure on the first evening. The fortress-like building was founded in 785. Entering from a superb courtyard of orange trees, inside are endless rows of two-tiered arches in stripes of red brick and white stone, supported on 850 columns. These become especially elaborate around the mihrab (prayer niche). Strangely in its centre is a cathedral, began when the Christians took over in 1236. I really don't think that planning permission would be granted these days! It was highly controversial even then. The tour was followed by a welcome reception at the Christian King's Palace (the Alcazar dates from C13) and a superb acoustic guitar recital. Tapas at around 11pm just about saved the lives of those North Americans who usually eat at 6pm.The second evening was yet another reception at Circulo de la Amistad restaurant. The preservation and beauty of these surroundings continued to amaze.

Wednesday saw an early start to visit an olive oil processing factory in Baena town, past endless stands of olive trees on rolling hills interspersed with the characteristic white villages. The family and parts of the mill and cellars have been in place for 200 years. High quality oil is produced by traditional means from organically grown, hand-picked olives. The enthusiastic manager explained the different qualities of the pickings: from piquant in mid November to sweeter and blander in the New Year. Three main varieties are blended to combine "flowery" aroma from one to stability conferred by high poly-phenols from another variety. There we had a remarkable breakfast, based on a "brunch" shared by the late night shift workers and the incoming millers. Traditional bread, baked in a wood fired oven, drizzled with oil, was accompanied by ham, cheese, salted cod and oranges. In particular the oil was superb; it was highly turbid and is collected by partial cold extraction  before even the cold press. Add quality rioja and you have a roomful of mellow pathologists, all before 10 am. 

Breakfast in the olive oil factory. Rioja begins to take effect. Deb Fravel (Beltsville, USA), 
Rafael Jiminez, the symposium organiser (Cordoba), George Lazarovits (Ontario, Canada), Bud Platt (PEI, Canada). 

Next was the Gracia wine cellar in nearby Montilla, a centre for Cordoba's sherry-like wines. They range from dry fino (check this very one out in UK Tescos) through to sweet, dark Pedro Jiminez. We had a fascinating in depth tour of the combination of modern fermentation technology and traditional maturation in oak barrels. Tasting was obligatory. Also it got us in the mood for lunch at a Montilla restaurant. This was sensory overload!

The final evening was spent at the renowned Bodegas Campos restaurant. Fine food and wine was followed, inevitably, by flamenco. This was excellent until certain pathologists were invited to contribute; there were some pretty good attempts, but most should stick to PCR or whatever they do best.

Health warning We wish to thank BSPP for support to attend this meeting. Will BSPP also subsidise a diet and exercise plan to get us back into shape? The public just don't realise the risks we have to take at these conferences. Also at least three of the UK delegates are likely to suffer back injury as slabs are ripped up, concrete smashed, decking removed and soil levelled in order to reproduce a Cordoban patio complete with pebble mosaics and a central fountain.

Richard M. Cooper, University of Bath
Dimitra Gkilpathi, IACR-Rothamsted
Tom Locke, ADAS