Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.     

Pathogenesis ofSynchytrium endobioticum VII. Earthworms as vectors of wart disease of potato

Zoospore motility favoured infection when sprouts and inoculum source were contigous. Infection from more distant inoculum involved soil fauna; earthworms were associated with infected plants 9 to 25 cm away from the inoculum source.Contribution No 93, Research Station, Agriculture Canada, P. O. Box 7098, A1E 3Y3.     

Pathogenesis ofSynchytrium endobioticum

Summary Field plots infested withSynchytrium endobioticum pathotype 2 were amended with agricultural lime, NH₄NO₃ and/or urea and planted with cv. Arran Victory. A weekly survey of changes in pH from planting to harvest revealed constant pH in absence of the chemicals, fluctuating pH with lime, very large increase then falling off with urea, and depressed below normal pH with NH₄NO₃. Best disease control occurred in urea-treated plotsvs NH₄NO₃-treated plots. Addition of lime, although it elevated pH, did not suppress the disease with urea or NH₄NO₃ added. Mechanisms for suppression by urea are related to its role as an ammonia producer and microbial stimulator, and their implication for infection byS. endobioticum.Contribution No. 81, Research Station, Agriculture Canada, P.O. Box 7098 St. John's, Newfoundland, A1E 3Y3     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

A quantitative method to examine large numbers of soil samples for Synchytrium endobioticum,the cause of potato wart disease

Summary Resting sporangia were collected from dried soil on 37-m mesh, extracted with chloroform, and deposited on grid, marked membrane filters. The technique gives consistent figures, a rapid estimate of the inoculum potential of a soil, and is suited for analysis of large volumes of soil samples.Contribution No. 47, Research Station, Agriculture Canada, P.O. Box 7098, St. John's Newfoundland.     

Patterns of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot disease of pears, in relation to weather conditions

Seven-day volumetric spore samplers were installed in pear orchards of northern Italy, in the years between 1993 and 2002, and operated continuously during the development of brown spot epidemics (mid-April–mid-August), caused by Stemphylium vesicarium. Aerial concentration of conidia was recorded at 2 h intervals to study their diurnal and seasonal patterns and the influence of weather conditions. The diurnal periodicity of aerial conidia showed a peak around midday and low counts in the dark. The increase in spore concentration was significantly correlated with the reduction of relative humidity and wetness in early morning, and the increase of wind in late morning and afternoon. Conidia of S. vesicarium became easily airborne to form a regular component of the air-spora in pear orchards, while ascospores were caught only sporadically. Differences between years concerned total spore counts and numbers of peaks (defined as days with more than 30 conidia/m³ air per day). Periods with highest spore counts occurred in late-May to early-June (in 2 years), mid to end of June (5 years), or after mid-July (3 years). There was a significant correlation between spore peaks and days with favourable weather conditions, defined as days with air temperature between 15 and 25°C and high humidity, particularly a wet period longer than 10 h. Occurrence of one or more consecutive days with favourable weather conditions determined an increase in the airborne concentration of conidia, which usually lasted some days and then decreased.     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Pine wilt disease as promising causal agent of the mass mortality of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima in the field

The causal agent of the mass mortality of field populations of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima (PAA) was investigated with special respect to the involvement of pine wilt disease. Wood chips, branches and/or increment cores for detecting the pinewood nematode, feeding marks of the vector insect and environmental stress in the past, respectively, were taken from live and dead PAA trees grown in three locations, Yaku-shima and Tanega-shima Islands and a plantation in Kagoshima City, from 1997 to 1998. Five trees died after the spring of 1996 and, of these, four were inhabited by the pinewood nematode. Feeding marks of the vector insects were found on the branches of all dead trees and most of the live trees investigated. These results suggest that the infection of pine wilt disease in PAA trees occurs in the field. Annual ring growth of the sample trees showed neither intervention nor growth reduction, which implies strong environmental stress that may cause mortality in PAA trees.     

QTL analysis of potato tuberization

Quantitative trait loci (QTLs) affecting tuberization were detected in reciprocal backcrosses between Solanum tuberosum and S. berthaultii. Linkage analyses were performed between traits and RFLP alleles segregating from both the hybrid and the recurrent parent using a set of framework markers from the potato map. Eleven distinct loci on seven chromosomes were associated with variation in tuberization. Most of the loci had small effects, but a QTL explaining 27% of the variance was found on chromosome 5. More QTLs were detected while following alleles segregating from the recurrent S. tuberosum parent used to make the backcross than were detected by following alleles segregating from the hybrid parent. More than half of the alleles favoring tuberization were at least partly dominant. Tuberization was favored by an allele from S. berthaultii at 3 of the 5 QTLs detected by segregation from the hybrid parent. The additive effects of the QTLs for tuberization explained up to 53% of the phenotypic variance, and inclusion of epistatic effects increased this figure to 60%. The most common form of epistasis was that in which presence of an allele at each of 2 loci favoring tuberization was no more effective than the presence of a favorable allele at 1 of the 2 loci. The QTLs detected for tuberization traits are discussed in relationship to those previously detected for trichome-mediated insect resistance derived from the unadapted wild species.Paper number 54 of the Department of Fruit and Vegetable Science, Cornell University     

The effect of biofungicide Mycostop on Ceratocystis radicicola, the causal agent of black scorch on date palm

In vitro studies were conducted to assess the efficacy of the biofungicide Mycostop on the fungus Ceratocystis radicicola (= Ceratostomella radicicola), which causes black scorch on date palm (Phoenix dactylifera) and the effect salinity on the growth of Streptomyces griseoviridis, the activecomponent of Mycostop. C. radicicola wasisolated from roots of diseased date palm inlandscape settings, nurseries, andplantations. In vitro, the effectiveconcentration (EC₅₀) of Mycostop thatreduced the mycelial growth by 50% was 0.35 g/l of PDA. Mycostop at the rate of 0.35 g/lor greater reduced spore germination,plasmolyzed germlings and reduced sporulationof C. radicicola. In essence it reducedthe inoculum potential of C. radicicola. In sterile soil treated with Mycostop, rootpieces of date palm inoculated with C.radicicola were less necrotic than those inuntreated soil. Streptomycesgriseoviridis in Mycostop did not grow on PDAamended with 5.0% NaCl (–4.28 MPa), but thenumber of colony formed units on PDA amendedwith 1.0 to 4.0% NaCl was not significantlydifferent from a factor that was considered ifMycostop is to be used to manage black scorchin Kuwait. The observations showed the effectsof Mycostop on C. radicicola inlaboratory studies but its efficacy on blackscorch disease of date palms in nurseries andplantations is yet to be determined.     

QTL analysis of potato tuber dormancy

The potential loss of chemical sprout inhibitors because of public concern over the use of pesticides underscores the desirability of breeding for long dormancy of potato (Solanum tuberosum L.) tubers. Quantitative trait locus (QTL) analyses were performed in reciprocal backcrosses between S. tuberosum and S. berthaultii toward defining the complexity of dormancy. S. berthaultii is a wild Bolivian species characterized by a short-day requirement for tuberization, long tuber dormancy, and resistance to several insect pests. RFLP alleles segregating from the recurrent parents as well as from the interspecific hybrid were monitored in two segregating progenies. We detected QTLs on nine chromosomes that affected tuber dormancy, either alone or through epistatic interactions. Alleles from the wild parent promoted dormancy, with the largest effect at a QTL on chromosome 2. Long dormancy appeared to be recessive in the backcross to S. berthaultii (BCB). In BCB the additive effects of dormancy QTLs accounted for 48% of the measured phenotypic variance, and adding epistatic effects to the model explained only 4% more. In contrast, additive effects explained only 16% of the variance in the backcross to S. tuberosum (BCT), and an additional 24% was explained by the inclusion of epistatic effects. In BCB variation at all QTLs detected was associated with RFLP alleles segregating from the hybrid parent; in BCT all QTLs except for two found through epistasis were detected through RFLP alleles segregating from the recurrent parent. At least three dormancy QTLs mapped to markers previously found to be associated with tuberization in these crosses.Paper number 55 of the Department of Fruit and Vegetable Science, Cornell University     

Three newAspergillus species isolated from clinical sources as a causal agent of human aspergillosis

Three new species ofAspergillus isolated from clinical sources in China are described and illustrated:A. beijingensis, A. qizutongii andA. wangduanlii. The first species is characterized by spreading colonies, yellow to grayish green conidial heads, smooth-walled conidiophores with a clavate vesicle, uniseriate aspergilla and nearly globose, micro-verrucose conidia. The second is characterized by spreading colonies, olive-yellow conidial heads, conspicuously roughened conidiophores with a flask-shaped vesicle, uniseriate but often secondarily proliferating aspergilla and globose, smooth conidia. The third is characterized by rapidly growing colonies, dull green conidial heads, smooth to irregularly roughened conidiophores which are often surrounded by coiled hyphae in the basal part and are terminally swollen into a globose or irregular shaped vesicle, uniseriate aspergilla and globose, micro-verrucose conidia.     

Progeny tests of barley,wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance

Summary Because plant cells cultured in vitro express genetic variability and since they can be regenerated into functional plants, procedures have been designed to use this system for the production of plants with new important agronomic characteristics, particularly for disease resistance. For barley, wheat, and potato somaclones have been found that were less susceptible to a toxin of Helminthosporium, fusaric acid, Fusarium coeruleum, F. sulphureum, or Phytophthora infestans, when screened in the first in-vitro-derived generation. Here the progeny of such somaclones is evaluated after natural and artificial infection, using greenhouse-grown or field material. The progenies of the same somaclones did not express detectable differences, which indicated that no heterozygous mutations occurred. Most lines and clones differed in their level of susceptibility to the pathogen compared to the level of the starting material, but these data were in no instance significant. It is discussed here whether this lack of significance is due to a lack of genetic differences or whether the test procedures are in adequate for detecting and securing the slight, probably quantitative, alterations.     

Isozyme and plastid DNA assessment of pedigrees of nineteenth century potato cultivars

Summary Isozyme and ctDNA RFLP patterns were determined for ten historically important potato cultivars (Solanum tuberosum ssp. tuberosuni) in order to relate and confirm their pedigrees. Isozyme polymorphism was detected at 11 of 13 loci examined, whereas only T-type cytoplasm, the predominant ctDNA of S. tuberosum ssp. tuberosum, was observed. Isozyme analysis indicated that potato cultivars previously presumed to be derived from open-pollinated berries of Garnet Chili and Early Rose were in fact the result of hybridizations. In addition, putative pedigrees of Irish Cobbler, White Rose, and Bliss Triumph were not supported. Garnet Chili, the first derivative of Rough Purple Chili, carries allozmyes at Mdh-1 and Pgm-2, which supports the Chilean origin of Rough Purple Chili. The identical ctDNA pattern among the cultivars may indicate a common maternal lineage that traces through Garnet Chili to Rough Purple Chili. The allozyme frequencies estimated from these cultivars provide a base from which subsequent introductions of Solanum species into the ssp. tuberosum gene pool can be assessed.Journal Article No. 000123