Kenneth L. Deahl

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Identification of mating types and metalaxyl resistance in North American populations ofPhytophthora infestans

The A2 mating type ofPhytophthora infestans was first reported in the United States in 1990. Concurrently,P. infestans strains resistant to metalaxyl ere found in the Pacific Northwest. Collaborative surveys were undertaken during 1991–1993 to investigate the frequency of occurrence of A2 mating types and metalaxyl resistant strains in populations ofP. infestans isolated from outbreaks of late blight in potato and tomato crops in North America.In vitro testing indicated that isolates from the northeastern U.S. and Atlantic Canada were primarily (52/55) metalaxyl sensitive and all were A1 mating types. Among 85 isolates from late blight epidemics in Florida and Texas, greater than 61% were both metalaxyl resistant and A2 mating type. Metalaxyl resistance and A2 mating types were identified also in a few tomato isolates from North Carolina. Although the majority of 134 isolates from the Pacific Northwest (British Columbia and Washington) were metalaxyl resistant, only 2 isolates from Washington were A2 mating types. Among 111 isolates from 2 sites in central Mexico, 63% and 77% were both metalaxyl resistant and A2 mating types. The data indicate also a higher frequency of metalaxyl resistance in A2 isolates, than in A1 isolates, among isolates from Florida and Texas. Highest metalaxyl resistance levels were found, however, in A1 isolates from California, where no A2 isolates were recovered.CompendioEl tipo reproductivo A2 de Phytophthora infestans fue reportado por primera vez en los Estados Unidos en 1990. Simultáneamente, se encontraron en el noroeste del Pacífíco variantes de P infestans, resistentes al metalaxyl. Durante 1991–1993, se llevaron a cabo encuestas colaborativas para investigar la frecuencia conque se presentan los tipos A2 y las variantes resistentes al metalaxyl en las poblaciones de P. infestans aisladas, en las epidemias de tizón tardío, de los eultivos de papa y tomate en Norteamérica. La pruebas in vitro indicaron que los aislamientos del noreste de los Estados Unidos y de la zona canadiense del Atlántico fueron principalmente (52/ 55) sensibles al metalaxyl, perteneciendo todos ellos al tipo reproductivo A1. Entre 85 aislamientos de epidemias de tizón tardío en Florida y Texas, m as de 61 % fueron resistentes al metalaxyl y del tipo reproductivo A2. Se identificaron también la resistencia al metalaxyl y los tipos reproductivos A2 en algunos aislamientos de tomate en Carolina del Norte. Aunque la mayoría de 134 aislamientos del noroeste del Pacífico (Columbia Británica y Washington) fueron resistentes al metalaxyl, sólo dos aislamientos de Washington fueron de los tipos reproductivos A2. Entre 111 aislamientos de dos lugares del centro de México, 63% y 77% fueron tanto resistentes al metalaxyl como de los tipos reproductivos A2. Los datos también señalan una mayor frecuencia de resistencia al metalaxyl en los aislamientos A2 que en los A1 efectuados en Florida y Texas. Sin embargo, se encontraron niveles más altos de resistencia al metalaxyl en los aislamientos A1 de California, donde no se recuperaron aislamientos A2.

Recent genotypes of Phytophthora infestans in the eastern United States reveal clonal populations and reappearance of mefenoxam sensitivity.

Isolates of Phytophthora infestans (n = 178) were collected in 2002 to 2009 from the eastern United States, Midwestern United States, and eastern Canada. Multilocus genotypes were defined using allozyme genotyping, and DNA fingerprinting with the RG-57 probe. Several previously described and three new mulitilocus genotypes were detected. The US-8 genotype was found commonly on commercial potato crops but not on tomato. US-20 was found on tomato in North Carolina from 2002 through 2007 and in Florida in 2005. US-21 was found on tomato in North Carolina in 2005 and Florida in 2006 and 2007. US-22 was detected on tomato in 2007 in Tennessee and New York and became widespread in 2009. US-22 was found in 12 states on tomato and potato and was spread on tomato transplants. This genotype accounted for about 60% of all the isolates genotyped. The US-23 genotype was found in Maryland, Virginia, Pennsylvania, and Delaware on both tomato and potato in 2009. The US-24 genotype was found only in North Dakota in 2009. A1 and A2 mating types were found in close proximity on potato and tomato crops in Pennsylvania and Virginia; therefore, the possibility of sexual reproduction should be monitored. Whereas most individuals of US-8 and US-20 were resistant to mefenoxam, US-21 appeared to be intermediately sensitive, and isolates of US-22, US-23, and US-24 were largely sensitive to mefenoxam. On the basis of sequence analysis of the ras gene, these latter three genotypes appear to have been derived from a common ancestor. Further field and laboratory studies are underway using simple sequence repeat genotyping to monitor current changes in the population structure of P. infestans causing late blight in North America.

Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora.

Twenty-one homologs of family 5 endo-(1–4)-β-glucanase genes (EGLs) were identified and characterized in the oomycete plant pathogens Phytophthora infestans, P. sojae, and P. ramorum, providing the first comprehensive analysis of this family in Phytophthora. Phylogenetic analysis revealed that these genes constitute a unique eukaryotic group, with closest similarity to bacterial endoglucanases. Many of the identified EGL copies were clustered in a few genomic regions, and contained from zero to three introns. Using reverse transcription PCR to study in vitro and in planta gene expression levels of P. sojae, we detected partially processed RNA transcripts retaining one or more of their introns. In some cases, the positions of intron/exon splicing sites were also found to be variable. The relative proportions of these transcripts remain apparently unchanged under various growing conditions, but differ among orthogolous copies of the three Phytophthora species. The alternate processing of introns in this group of EGLs generates both coding and non-coding RNA isoforms. This is the first report on Phytophthora family 5 endoglucanases, and the first record for alternative intron processing of oomycete transcripts.

MONITORING THE RATE OF OXYGEN CONSUMPTION IN PLANT CELL SUSPENSIONS

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered.

Characteristics of Phytophthora infestans isolates from Uruguay

Isolates of Phytophthora infestans were obtained from late blighted plants from several potato-growing regions of Uruguay in 1998 and 1999. Of these, 25 representative isolates (4 from 1998, 21 from 1999) from the main potato-growing areas of the country, were characterised in terms of mating type, metalaxyl resistance, allozyme genotype, mitochondrial haplotype, RG57 fingerprint (1999 isolates only) and pathotype. All isolates proved to be A2 mating type, monomorphic and homozygous at the loci coding for glucose-6-phosphate isomerase and peptidase (Gpi 100/100, Pep 100/100) and to possess mitochondrial haplotype IIa. Metalaxyl-resistant isolates constituted 92% of the total. All the 1999 isolates possessed the same RG57 fingerprint, which was that previously reported as associated with the clonal lineage BR-1 from Brazil and Bolivia, which is also A2, Gpi 100/100, Pep 100/100. Most of the isolates displayed broad-spectrum virulence and five carried virulence to 10 of the 11 R genes tested despite the absence of R genes in commercially grown potato cultivars. It was concluded that the Uruguayan P. infestans isolates resembled isolates from neighbouring South American countries, notably Brazil, and belong to the new populations of the pathogen now predominant in many countries.

Differential neurosensory responses of adult Colorado potato beetle, Leptinotarsa decemlineata, to glycoalkaloids.

Neurons from chemosensory hairs on the galeae of adult Colorado potato beetle (CPB), Leptinotarsa decemlineata(Say), were investigated for responses to glycoalkaloids of the family Solanaceae. While solanine and tomatine elicited irregular firing by multiple neurons and bursting activity at 1 mM concentration in most sensory hairs, stimulation with leptine I resulted in consistently high-frequency, slowly adapting responses with a dose-dependent effect between 0.03 and 0.3 mM concentrations. Responses to a mixture of solanine and leptine I suggested possible modification of the leptine I response by other glycoalkaloids, resulting in reduced neural activity relative to leptine I alone. These results establish a method for specifically evaluating leptine I and other glycoalkaloids for effects on feeding behavior of CPB and provide a sensory component for incorporating deterrent chemistry into biorational control methods for the CPB.

Effect of α-tomatine and tomatidine on the growth and development of the Colorado potato beetle (Coleoptera: Chrysomelidae): studies using synthetic diets.

Glycoalkaloids are found throughout the genera Solanum (potato) and Lycopersicon (tomato). Certain glycoalkaloids, i.e., α-tomatine, solanocardenine, and leptine, have been implicated as resistance...

Natural Occurrence of Phytophthora infestans on Black Nightshade (Solanum nigrum) in Wales

There is only one published record of natural infection of black nightshade (Solanum nigrum L.) by Phytophthora infestans (Mont.) de Bary in England (3) and none from Wales. In August 2001, brown, necrotic leaf lesions with pale green margins were found on black nightshade weeds in a potato trial naturally infected with P. infestans at Henfaes Research Centre, University of Wales, Bangor. Although the plants were low growing with large, succulent leaves 4 to 5 cm long instead of having a more erect habit and smaller leaves, their identity was confirmed as S. nigrum; their atypical appearance may relate to the known phenotypic plasticity of this species (4). Infected leaflets incubated in moist chambers produced sporangia typical of P. infestans, and zoospores were released after chilling in water. Five isolates obtained from leaf fragments had growth on rye agar that was indistinguishable from that of P. infestans from potato. Detached leaflets of S. nigrum and S. tuberosum cv. Green Mountain inoculated with the S. nigrum isolates developed sporulating lesions under high humidity in 7 to 10 days; uninoculated controls remained symptomless. Inoculation of attached leaves of 10 potted S. nigrum plants resulted in seven plants developing necrotic lesions with a few sporangia 10 to 14 days later; sporulation developed mainly on lower leaves of plants that were older or had senesced. The remaining plants developed necrotic lesions with no sporulation, and P. infestans was reisolated from sporulating and nonsporulating lesions. All isolates were A1 mating type, metalaxyl-sensitive, and mitochondrial haplotype IIa, which are characteristics found commonly in isolates of P. infestans from potato in Wales (1). Single-sporangial isolates from each isolate were homozygous for glucose-6-phosphate isomerase and peptidase (Gpi 100/100, Pep 100/100). RG57 fingerprint analysis further established that all five black nightshade isolates were identical to each other and to some local P. infestans isolates from potato. P. infestans in Wales belongs to the new population (1), which may infect a wider host-range than the old US-1 clonal lineage. However, infected black nightshade was only found after late blight was widespread in potato fields. In subsequent years at the same site, weeds of S. nigrum have remained noninfected despite high levels of late blight pressure on adjacent potato plots. There is no evidence to suggest that this species acts as an overwintering host in Wales since it is an annual and lacks frost resistance. Field infection of S. nigrum by P. infestans has recently been reported in the Netherlands (2). Our observations confirm the potential of P. infestans to infect another solanaceous plant species. Alternative hosts may interfere with current disease control strategies because infected weeds would escape fungicide application and could serve as reservoirs of inoculum throughout the growing season. References: (1) J. P. Day and R. C. Shattock. Eur. J. Plant Pathol. 103:379, 1997. (2) W. G. Flier et al. Plant Pathol. 52:595, 2003. (3) J. M. Hirst and O. J. Steadman. Ann. Appl. Biol. 48:489, 1960. (4) B. S. Rogers and A. G. Ogg Jr. Page 30 in: Biology of Weeds of the Solanum Nigrum Complex (Solanum Section Solanum) in North America. USDA Publication ARM-W-23, 1981.

Gene silencing indicates a role for potato endoglucanase inhibitor protein in germplasm resistance to late blight

Durable resistance to late blight is believed to be dependent on genes other than R genes. Reduced rates of lesion expansion, as found in the germplasm B 0767-2, can provide durable resistance by limiting tissue damage and reducing pathogen populations. We have tested the role of the proteinaceous xyloglucan-specific endoglucanase inhibitor (XEIP) in limiting late blight lesion expansion in B 0767-2. Plant material was silenced for the XEIP using localized infiltration ofAgrobacterium tumefaciens harboring pCAMBIA 1302 containing an antisense copy of XEIP. Plant cell transformations with this construct induced effective silencing as indicated by a reduction of up to 80% in XEIP transcript levels. Silencing of XEIP resulted in a marked increase in lesion size and water soaking, indicating that the inhibitor protein plays a role in limiting lesion expansion in B 0767-2.ResumenSe cree que la resistencia durable al tizón tardío es dependiente de genes diferentes a los genes R. Tasas reducidas de expansión de las lesiones como las encontradas en el germoplasma B 0767-2 pueden proporcionar resistencia durable, limitando el daño al tejido y reduciendo la población del patógeno. Hemos evaluado el rol del inhibidor endogluconasa xyloglucan proteinaceo específico (XEIP) para limitar la expansión de la lesión de tizón tardío en B 0767-2. El material vegetal quedó silenciado para el XEIP por infiltración localizada deAgrobacterium tumefaciens produciendo pCAMBIA 1302 que contiene una copia de XEIP en sentido contrario. Las transformaciones celulares de la planta con este “construct” indujeron un silenciado efectivo, indicado por una reducción de hasta en un 80% en los niveles de trascripción del XEIP. El silenciamiento del XEIP dio como resultado un marcado crecimiento del tamaño de la lesión y la absorción de agua, indicando que la proteína del inhibidor limita la expansión de la lesión en B 0767-2.