The aims of the project were to define the signals provided by the cereal host which induce cereal rusts to form appressoria over stomata, and to analyse the mechanism by which these signals are transduced. Using polystyrene replicas of microfabricated silicon wafers to provide precisely defined topographies, appressoria were induced in all the cereal rusts analysed (<I>Puccinia graminis</I> f. sp. <I>tritici, P. graminis </I>f. sp. <I>avenae, P. graminis </I>f. sp. <I>secalis, P. recondita</I> f. sp. <I>tritici, P. coronata </I>f. sp. <I>avenae </I>and <I>P. hordei</I>) by topographies with closely spaced (1.5 μm) ridges. Ridges of different heights (between 0.116 μm and 2.4 μm) were tested. The patterns of response to different ridge heights was not correlated to host nor rust species. Only one rust, <I>P. graminis</I> f. sp. <I>secalis</I>, formed significant numbers of appressoria (up to 62%) on ridges spaced 50 μm apart (= single ridges). The formation of appressoria on host leaves was found to be very efficient, 92-95% of germlings encountering a stoma formed an appressorium over the stoma. On polystyrene host leaf replicas, however, the efficiency was much less, and varied significantly between rusts (4%-63%). Again, the variation was not correlated to host or rust species. The implication was that while topographical signals alone are sufficient to induce appressoria <I>in vitro</I>, topography of the host alone was not sufficient to explain the high efficiency of appressorium induction seen <I>in vivo</I>. It was shown for <I>P. graminis </I>f.sp<I>. tritici</I> that the volatile compounds <I>trans</I>-2-hexen-1-ol and <I>cis</I>-3-hexen-1-ol, produced by the host plant as part of the lipoxygenase pathway, induced appressoria in the absence of topographical stimuli. Induction was dose dependent and optimal at 1 mM when applied to the fungus as a solution. Appressoria were also induced when the germlings were exposed to vapour from solutions of <I>trans</I>-2-hexen-1-ol. Induction was over a very narrow range of concentrations (1-2 mM).

No description available.

Soil samples were incubated on agar precolonised by different 'host' fungi for detection of presumptive mycoparasites. In a survey of 34 British soils, only <I>Pythium oligandrum, Gliocladium roseum</I> group, <I>Trichoderma</I> spp, and <I>Papulaspora</I> sp. were detected routinely, from 19, 34, 30 and 27 soils respectively; all soils contained more than one mycoparasite, 3 soils had two detectable types, 20 contained three types and 11 contained all four detectable types. The choice of host fungus strongly influenced the efficiency of detection of mycoparasites: <I>Fusarium culmorum</I> was best for <I>P. oligandrum, Rhizoctonia solani</I> for <I>Trichoderma</I> spp., and <I>Botrytis cinerea</I> for <I>Papulaspora</I> sp., but <I>G. roseum</I> (and <I>G. atrum</I> and <I>G.fimbriatum</I>) grew well from soil on colonies of <I>Trichoderma aureoviride</I>, <I>R. solani</I> or <I>B. cinerea</I>. The incidence of detection in replicate samples indicated that a range of host-colonised agar plates are needed to determine the spectrum of presumptive mycoparasites in a soil. Competition between these mycoparasites affected the success of detection because incorporation of metalaxyl into <I>B. cinerea</I>-colonised agar plates prevented growth by <I>P. oligandrum</I> and enhanced the growth of <I>Papulaspora</I> sp. from soil samples. Baiting of soils only partly increased the efficiencies of detection: for <I>Trichoderma</I> spp, when soil was baited with cellulose film precolonised by <I>R. oryzae, </I>and for <I>Papulaspora</I> sp. when the bait was cellulose film or mature, dried wheat leaves, precolonised by <I>Humicola grisea.</I> Agar precolonised by <I>F. culmorum</I> was used to detect apparent changes in soil populations of <I>P. oligandrum</I>, using a most probable number method based on serial dilution of soil samples with sand. The addition of dried, mature wheat flag leaves or fresh, green grass leaves to soil enhanced the detectable populations for at least 280 days, although the effect of grass leaves was found only after a second supplement at 150 days. A metalaxyl-tolerant population of <I>P. oligandrum</I>, added to soil as oospores, was still detectable after 240 days when soil samples were placed on <I>F. culmorum</I>-colonised agar containing metalaxyl.

In the first part of this thesis, the developmental strategies of the two contrasting barley leaf rusts, <I>Puccinia striformis</I> f. sp. <I>hordei</I> (barley yellow rust, BYR) and <I>P. hordei</I> (barley brown rust, BBR) were compared during infection of the susceptible cultivar Golden Promise. The techniques of fluorescence microscopy and low-temperature scanning microscopy were used correlatively to provide complementary information on the temporal and spatial development of these rusts within the host. These studies revealed many differences between the two rusts, including: (1) the germ tubes of BYR were long and unbranched whilst those of BBR were short and branched. (2) Only BBR produced appressoria. (3) The spread from the substomatal cavity into surrounding leaf tissue was delayed in BYR but not BBR. (4) BYR formed large, aseptate, invasive runner hyphae prior to reproduction after which they became septate. BBR lacked runner hyphae. (5) BYR exhibited semi-systemic growth during the colonisation of leaf tissue whilst BBR colonised leaf tissue only around the site of penetration. (6) BYR produced morphologically distinct hyphae involved in uredinal bed formation whilst BBR did not. (7) Primary uredina were formed some distance from the site of penetration by BYR whilst in BBR, uredinia formed at the site of penetration. In the second part of this study, quantitative comparisons were made of the contrasting infection strategies of the two rusts in their respective optimum conditions for development. This study revealed for the first time that the semi-systemic infection strategy employed by BYR was more efficient than the localised infection strategy exemplified by BBR. The final part of the investigation involved a study of the role of contact sensing in the induction of appressorium formation by <I>P. hordei</I> and <I>P. graminis</I> f. sp. <I>tritici</I> (wheat stem rust). Polystyrene replicas of microfabricated silicon wafers, bearing precisely defined topographies, were used as artificial substrata for growing these cereal rust <I>in vitro</I>.

No description available.

The existence, nature and possible functions of double-stranded RNA (dsRNA) was studied in strains of <I>Rhizoctonia solani</I> anastomosis group (AG) 3, which infects potatoes, and in some other AGs of <I>R. solani. </I>The aim was to determine whether dsRNA might be exploited as a basis for reducing the virulence of <I>R. solani</I> strains, as occurs in <I>Cryphonectria parasitica,</I> a pathogen of chestnut trees. Isolates of <I>R. solani </I>AG 3 were obtained from potato tubers from a single field site, and from geographically distant sites. DsRNA was found to be ubiquitous, with multiple elements present in each strain, as determined by CF11 cellulose chromatography. Similar gel banding patterns were observed between strains isolated from separate tubers within a single field site; however, banding patterns differed between isolates from diverse sources. All the AG 3 isolates were assessed as being weakly virulent in seedling assays on six host crops (carrot, cress, lettuce, onion, radish, tomato). Attempts to "cure" strains of dsRNA by repeated hyphal tip, subculturing or by growing strains in the presence of cycloheximide were generally unsuccessful; although some individual dsRNA bands were lost, they sometimes reappeared, potentially indicating the presence of a chromosomally, integrated copy of the dsRNA. Partially cured strains were unaltered in virulence compared with their respective parental strains. To determine whether dsRNA elements might be transmitted throughout field populations by hyphal anastomosis, strains were paired in various combinations on agar and examined microscopically. Strains from single tubers were compatible with one another; but isolates from different tubers showed a high degree of incompatibility with one another, and isolates from separate fields were incompatible with each other.

A reverse transcription-polymerase chain reaction (RT-PCR) assay was shown to be sensitive and reliable for detection of potato mop-top virus (PMTV) RNA sequences in the flesh of infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferous <I>Spongospora subterranea </I>in agricultural soils, and of PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional bait-tests and sap inoculation methods for the detection and diagnosis of PMTV. PMTV was detected by ELISA in primary zoospores from four out of six isolates of <I>S. subterranea </I>f. sp. <I>subterranea. </I>One virus-free isolate (N) of <I>S. subterranea </I>was used to acquire PMTV from potato roots and to transmit the virus to healthy plants. A mono-fungal culture of <I>S. subterranea </I>(isolate N) was derived by infecting tomato plant roots with a single cystosorus. The culture was used successfully to acquire PMTV from the roots of infected <I>Nicotiana debneyi </I>plants that had been manually inoculated with virus isolates, and subsequently to transmit the virus to test plants. These experiments confirm that <I>S. subterranea </I>is a vector of PMTV. Two PMTV isolates that had been maintained by manual inoculation for 19 and 21 passages were also acquired and transmitted by the fungus culture. A mono-fungal <I>S. subterranea </I>was unable to acquire and transmit PMTV-T which had ˜700 nucleotides deletion in the coat protein/ read-through (CP/RT) domain, while PMTV-S with a full-length CP/RT gene was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the 673 nucleotides of the 5'-end of RT gene of PMTV-S indicated that the difference of ˜700bp in RT gene of PMTV-T relative to that of PMTV-S occurs in 3'-half of the gene and is associated with loss of transmission by the fungus vector, <I>S. subterranea. </I>

The proteome of Peronospora viciae, an obligate biotrophic pathogen that causes downy mildew of pea (Pisum sativum), was investigated using 2D gel electrophoresis and mass spectrometry. This provided data on changes in protein abundance during the sequential development of pre-invasion stages, from un-germinated conidia to germinated conidia with germ tubes and appressoria. Preliminary work developed reproducible methods for in vitro germination of conidia through to appressorium development. The method developed for in vitro conidia germination resulted in up to 65% of germinated conidia, of whi ch 35% fonned an appressorium. Six methods for protein extraction from the pre-invasion stages were compared by ID and 2D SDS-PAGE electrophoresis. The method chosen had relatively simple and non -toxic constituents (urea, thiourea, CHAPS and Tris-HCl), and would be appropriate for use in a disposable diagnostic device. Gels of extracted proteins showed over 700 protein spots following Coomassie blue staining of 2D gels and over 2000 protein spots on difference gel electrophoresis (DIGE) gels. A total of 21 proteins were identified from P. viciae using MALDI - and Q-TOF mass spectrometry analysis and searching against the MASCOT databases. Of the identified proteins, the majority (2 Hsp 70, a succinate dehydrogenase, an enolase, a catalase, a S-adenosyl-L-methioninedependent methyltransferase, a Hsp72, 2 actins, three GAPDH, a Fructose-bisphosphate aldolase, a fatty acid phospholipid synthesis protein, an uncharacterized aldolase, a Protein CpnIO and a NDK) decreased in relative abundance during spore germination, but with no further change on fonnation of appressoria. In contrast, the relative abundance of calmodulin and chitin synthase remained constant throughout pre-invasion development. However, calmodulin was not detected using western blotting whereas isoforms of actin and GAPDH were detected. Data are interpreted in relation to P. viciae pre-invasion biology, and the identification of key proteins and biomarkers as novel targets for control, pathogen detection and disease diagnosis.

No description available.

No description available.