Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Carnation Fusarium wilt suppression in four composts

Fusarium wilt is now a major disease of carnation crops worldwide. Methyl bromide, which is used to remedy it, is environmentally unsafe. An alternative approach integrated into biological control is to grow crops in suppressive media. Suppressiveness of seven plant growth media to Fusarium oxysporum f. sp. dianthi was evaluated in bioassays with carnation (Dianthus cariophyllus) cv. Medea. These media were: (1) grape marc compost, (2) cork compost, (3) olive oil husk + cotton gin trash composted and mixed with rice husk, (4) spent mushroom compost mixed with peat, (5) coir fibre, (6) light peat and (7) vermiculite. In order to look for carnation Fusarium wilt suppressiveness indicators, growth medium pH and β-glucosidase activity were evaluated. Furthermore, F. oxysporum populations were measured in plant growth media at the beginning and end of bioassays. The compost media showed a range of suppressiveness in comparison with peat. Grape marc compost was the most effective plant growth medium in suppressing carnation Fusarium wilt. On the other hand coir fibre, peat and vermiculite were conducive for this disease. β-glucosidase activity and pH were positively correlated with disease severity as in other reports for tomato. Therefore, these two parameters are good indicators for carnation Fusarium wilt suppressiveness, and possibly for other F. oxysporum pathosystems. All composts showed similar F. oxysporum populations at the end of the bioassays to peat and vermiculite.     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Fusarium oxysporum f.sp. cucurbitacearum n.f. embracing all formae speciales of F. oxysporum attacking Cucurbitaceous crops

Isolates ofFusarium oxysporum from wilted muskmelons, watermelons, cucumbers and from the muskmelon rootstockBenincasa hispida were screened for pathogenicity on seedlings and adult plants of these crops and related species. In seedling tests the isolates were not typically species-specific, contrary to what might be expected as an implication of their characterization as forma specialis. They often attacked species of several genera of plants, but not beyond the family of theCucurbitaceae. In the adult stage, plants were much more exclusively attacked by their corresponding formae speciales, but essential exceptions occurred. Isolates from cucumber were highly pathogenic to muskmelons, in the adulstage even causing more wilt of the latter than of cucumber.Comparing the results of these experiments with data from the literature, it is argued that the proposed f.sp.cucurbitacearum, embracing all formae speciales which specialize on plants within the family of theCucurbitaceae, would best fit in with the present state of knowledge. A proposition is given for equivalence of old and new classifications of isolates.Samenvatting Isolaten vanFusarium oxysporum uit verwelkte meloenen, watermeloenen, komkommers en uit de meloene-onderstamBenincasa hispida werden getoetst op pathogeniteit voor zaailingen en volwassen planten van deze gewassen en verwante soorten. In zaailingtoetsen waren de isolaten weinig soort-specifiek, in tegenstelling tot wat mocht worden afgeleid uit hun karakterisering als forma specialis. Zij tastten vaak soorten uit verschillende geslachten aan, maar niet buiten de grenzen van de familie derCucurbitaceae. in het volwassen stadium waren de interacties veel specifieker en werden planten slechts aangetast door de bijbehorende formae speciales. Enkele essentiële uitzonderingen kwamen echter voor. Isolaten van komkommer waren zeer pathogeen voor meloen. In het volwassen stadium veroorzaakten zij zelfs sterkere verwelking van meloen dan van komkommer.Vergelijking van de resultaten van deze proeven met gegevens uit de literatuur leidt tot de conclusie dat de voorgestelde f.sp.cucurbitacearum, die alle formae speciales met specialisatie opCucurbitaceae omvat, het best overeenkomt met de huidige stand van kennis. Een voorstet wordt gedaan ter vervanging van de oude classificatie van isolaten door corresponderende nieuwe aanduidingen.     

Different ethylene production in vitro by several species and formae speciales of Fusarium

The ethylene production of severalFusarium species and formae speciales in vitro was measured under comparable conditions. All of them produced ethylene.Fusarium oxysporum Schlecht. ex Fr. f.tulipae Apt. produced much more than the other 18 strains investigated.Samenvatting Aangezien er aanwijzingen zijn dat ethyleen een rol speelt bij de aantasting van de tulpebol doorFusarium, werd de ethyleenproduktie van diverse soorten en formae speciales vanFusarium onder standaardvoorwaarden in vitro vergeleken. Alle onderzochteFusarium spp. en vormen vanFusarium produceerden ethyleen.Fusarium oxysporum Schlecht. ex Fr. f.tulipae Apt. bleek in vergelijking met 18 andere getoetste soorten en formae speciales enige duizenden malen meer ethyleen te produceren.     

Biofumigation with Brassica plants and its effect on the inoculum potential of Fusarium yellows of Brassica crops

The use of Brassica crops as green manure in the so-called biofumigation treatment has been successfully exploited for the management of soilborne pathogens and is gaining interest particularly in the case of less intensive agricultural systems. A study was undertaken to investigate possible negative side-effects of biofumigation in order to prevent possible damage caused by wilt pathogens able to attack both plants used for biofumigation as well as agricultural crops. To do so, firstly the response of different Brassicas, including some used in biofumigation, to the formae speciales of Fusarium oxysporum known for being pathogenic on Brassica crops was evaluated. Secondly, the effect of green manure treatments on yield, quality of crops, and inoculum densities, infection and survival of Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani was evaluated. In the second part of the work, four Brassica crops, selected for their response (susceptibility or resistance) to F. oxysporum f. sp. conglutinans and to F. oxysporum f. sp. raphani were evaluated in order to determine their response to the two pathogens during subsequent crops grown in soil where plants were incorporated as green manure into the soil at the end of each cycle. Moreover, the dynamics of the populations of F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani in the soil after several biofumigation cycles was studied. Many of the Brassica crops used for biofumigation tested were susceptible to F. oxysporum f. sp. conglutinans and or to F. oxysporum f. sp. raphani. Green manure treatment, carried out by growing nine cycles of biocidal plants, with a short crop cycle of 30–35 days, did not reduce Fusarium wilts on susceptible Brassica hosts. The population of the pathogen was partially increased as a result of the incorporation of tissues of the susceptible plants. When Brassica crops grown were resistant to the two F. oxysporum pathogens used for soil infestation, green manure simulation did inhibit both pathogens, thus confirming its biocidal activity. The results obtained under our experimental conditions show that biofumigation treatment is not applicable for soil disinfestation on crops susceptible to the same formae speciales of F. oxysporum affecting Brassica species used for biofumigation. Brassica crops resistant to Fusarium yellows should be grown where biofumigation is applied. Moreover, alternation of crops used for biofumigation should be encouraged.     

Genetic diversity and pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolated from wilted rocket plants in Italy

Thirty-six isolates of Fusarium oxysporum originated from Eruca vesicaria and Diplotaxis tenuifolia together with eight reference strains belonging to the formae speciales raphani, matthioli and conglutinans, typical on the Brassicaceae family, were tested for pathogenicity on two species of rocket plants (E. vesicaria L., syn. E. sativa, cv. ‘Rucola coltivata’; and D. tenuifolia cv. ‘Winter’) cultivated in the glasshouse. The results showed that different isolates were slightly, moderately or highly virulent. The strains were examined for differences in the nucleotide sequence of the ribosomal DNA (rDNA) intergenic spacer (IGS) region, about 2.5 kb long. The phylogenetic (neighbor-joining) analysis performed on the isolates enabled identification of four different groups, named I, II, III and IV. Thirty-one isolates out of 36 clustered in group I and were genetically similar to F. oxysporum f.sp. raphani. By considering the pathogenicity of the strains included in Group I, a partial host specialization could be observed: the average disease index of the isolates from D. tenuifolia was higher on wild rocket, whereas the average disease index of the isolates from E. vesicaria was higher on cultivated rocket. Moreover, isolates from cultivated rocket showed, on average, a higher degree of aggressiveness than the isolates from wild rocket. Concerning Group I, the sequence analysis confirmed the homogeneity of the population, with only five parsimony-informative SNPs and five haplotypes. Twenty-six out of 31 isolates belonged to haplotype 1. Groups II and III were genetically similar to strains of F. oxysporum f.sp. matthioli. Three other strains, not pathogenic or with a medium level of virulence, clustered together in Group 4, but their sequence was distant from that of other formae speciales. The pathogenicity and IGS analysis confirmed the presence of virulence variation and genetic diversity among the F. oxysporum isolates studied. To our knowledge, this is the first report of differentiation of formae speciales of F. oxysporum on rocket plants by IGS analysis.     

Extracts of killedPenicillium chrysogenum Induce resistance against Fusarium wilt of melon

Dry fungal biomass ofPenicillium chrysogenum (dry mycelium), a waste product of the pharmaceutical industry, was extracted with water and applied to the roots of melon plants before or after inoculation withFusarium oxysporum f.sp.melonis (Font). Seedlings (4–6 days after emergence) treated with either acidic dry mycelium extract (DME) or neutralized dry mycelium extract (NDME) were protected against challenge infection withFom. A single drench with 2–5% DME applied 12–72 h before inoculation provided significant control of the disease compared with water-drenched, challenged seedlings. No protection was seen in plants treated 0–6 h before inoculation or 0–48 h after inoculation. Neither DME nor NDME (0.5–5%) had any effect on fungal growthin vitro, which implied that disease controlin vivo was mediated by induced resistance. The resistance induced by DME protected melon plants not only against race 1,2, but also against the three other races of the pathogen, indicating a race-non-specific resistance againstFom. Both DME and NDME significantly increased peroxidase activity and free L-proline content in seedlings 12 h and 48 h after soil drench, respectively. Resistance to Fusarium wilt was significantly associated with elevated levels of peroxidase activity but not with free L-proline content. Thus, peroxidase might be involved in the defense mechanisms activated by DME or NDME. http://www.phytoparasitica.org posting Aug. 31, 2001.     

Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strains simultaneously colonizing root surfaces

 Green fluorescent protein (GFP)-marked Fusarium oxysporum f. sp. melonis and nonmarked F. oxysporum f. sp. fragariae were stained with neutral red. The neutral red stained vacuoles of the fungi without disturbing GFP fluorescence in the cytoplasm. GFP-marked fungi showed fluorescent hyphae with dark-stained vacuoles, whereas nonmarked fungi were detected as nonfluorescent hyphae with dark-dotted vacuoles. Root colonization by these two fungi was monitored using this method. Microconidia attached similarly to the root surface and elongated vegetative hyphae. Only the pathogenic fungi invaded, causing necrosis at the inoculation site. Thus, the present method enabled us to track simultaneously the various formae speciales of F. oxysporum colonizing the root surface. Received: March 25, 2002 / Accepted: September 27, 2002     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。     

Stem rot of jewel orchids caused by a new forma specialis,Fusarium oxysporum f. sp. anoectochili in Taiwan

Stem rot of Anoectochilus formosanus (Af) caused by Fusarium oxysporum (Fo) is a major limiting factor to jewel orchid production in Taiwan. Fo causes discoloration in vascular tissues. However, some newly collected Fo isolates from Af stem rot do not cause vascular discoloration, suggesting changes may have occurred in the pathogen. Among recent Fo isolates from Af there are two colony types, the cottony alba (CA) and the sporodochial (S). In order to confirm that both colony types cause Af stem rot, 200 isolates were obtained from diseased stems in Nantou County and characterized by colony type and whether or not the infected plants had vascular discoloration. Isolates of both the CA and S types caused stem rot of Af; some isolates in each colony type caused vascular discoloration whilst others did not. Pathogenicity tests with 22 isolates resulted in stem rot disease severity ratings on Af of 3·1–4·0 and 2·1–4·0 with CA and S type colonies, respectively. The same isolates failed to cause disease on Cattleya, Dendrobium or Phalaenopsis plants. Phylogenetic analysis of partial intergenic spacer sequences showed that these isolates were distinguishable from other formae speciales of Fo and could be separated into two groups correlated with the CA or S type colonies with high bootstrap. Based on pathogenic, morphological and molecular characterizations, the Fo that causes stem rot of Af is proposed to be a new forma specialis, F. oxysporum f. sp. anoectochili, with different pathotypes.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

The elegans fusaria causing wilt disease of carnation. II. Distinction of vegetative compatibility groups

The vegetative compatibility patterns among isolates ofElegans fusaria causing wilt disease of carnation were investigated. Nitrate non-utilizing mutants were generated from 16 isolates labelledF. redolens, nine of which came from carnation, and from 33 isolates labelledF. oxysporum, 19 of which came from carnation. Pairings of the mutants revealed five vegetative compatibility groups among the isolates from carnation, corresponding withF. oxysporum f.sp.dianthi race 1 (VCG1), race 2 (VCG2) and race 4 (VCG3),F. redolens f.sp.dianthi (VCG4) andF. redolens isolates from foot rot-diseased carnations (VCG5). Besides three isolates typical ofF. redolens, VCG4 comprised a now slightly deviating subculture of the type isolate ofF. redolens f.sp.dianthi of which the cultural characteristics correspond toF. oxysporum instead ofF. redolens. This observation may be taken to support previous conclusions that the distinction between both taxa is not justified. Otherwise, the compatibility patterns did not provide decisive evidence to accept or reject conspecificity of both taxa. Isolates from carnation did not form heterokaryons with other formae speciales ofF. oxysporum.Samenvatting De vegetatieve compatibiliteitspatronen bij isolaten vanElegans-fusaria die verwelkingsziekte bij anjer veroorzaken werden onderzocht. Van 16 isolaten vanF. redolens, waarvan negen afkomstig van anjers, en van 33 isolaten vanF. oxysporum, waarvan 19 afkomstig van anjers, werden mutanten gegenereerd die zonder een organische stikstofbron geen luchtmycelium meer konden vormen. Paringen tussen mutanten van isolaten afkomstig van anjers brachten een vijftal vegetatieve compatibiliteitsgroepen aan het licht, die overeenkwamen metF. oxysporum f.sp.dianthi fysio 1 (VCG 1), fysio 2 (VCG 2) en fysio 4 (VCG3),F. redolens f.sp.dianthi (VCG4) enF. redolens isolaten afkomstig van aan voetrot lijdende anjers (VCG5). Naast drie voorF. redolens karakteristieke isolaten omvatte VCG4 ook een afwijkende subculture van het type-isolaat vanF. redolens f.sp.dianthi, die in cultuureigenschappen overeen kwam metF. oxysporum in plaats vanF. redolens. Deze waarneming geeft enige steun aan eerdere conclusies dat het onderscheid tussen beide taxa niet gerechtvaardigd is. Daarbuiten gaven de compatibiliteitspatronen geen uitsluitsel over de mogelijke conspecificiteit van beide taxa. Isolaten afkomstig van anjers vormden geen heterokaryons met andere formae speciales vanF. oxysporum.     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.     

Inactivation of soil-borne plant pathogens during small-scale composting of crop residues

Samples of heavily infested crop residues were incorporated in static compost heaps (2.5–4.6 m³) of the Indore type. Temperature increased to 50–70°C within 6 days depending on the type of crop residues used and the location within the heap. The heat phase (>40 °C) lasted 2–3 weeks and was followed by a c. 5-months maturation phase (<40 °c).=" among=" the=" 17=" pathogens=" tested,=">Olpidium brassicae and one of the four formae speciales ofFusarium oxysporum that were tested survived composting, but also their inoculum was greatly reduced.Survival during specific phases of composting was studied by incorporation and retrieval of samples at various stages of the process.F. oxysporum f. sp.melonis was completely inactivated andO. brassicae andPlasmodiophora brassicae were almost completely inactivated during the short heat phase. The three pathogens survived the long-lasting maturation phase without loss of viability. Heat evolved during composting was found to be the most important factor involved with sanitation of crop residues. The possible involvement of fungitoxic conversion products and microbial antagonism is discussed.     

Fusarium wilt of common heliotrope (heliotropium europaeum)

Occurrence of Fusarium wilt of common heliotrope(Heliotropium europaeum L.) is reported. Pathogenicity of the causal agent was confirmed by reisolation from inoculated heliotrope plants. The specificity of the isolate was shown by cross-inoculation tests with five formae speciales and by inoculating the five respective hosts with a monoconidial isolate of the present fungus. Only the latter fungus was found to incite wilt in common heliotrope; the proposed name of this forma specialis isFusarium oxysporum f. sp.heliotropae nov. f.     

Suppression of Fusarium wilt of spinach with compost amendments

The effect of different organic composts on the suppression of wilt disease of spinach caused by Fusarium oxysporum f. sp. spinaciae was evaluated in a continuous cropping system in both containers and in microplot field trials. Test soils infested with the pathogen were amended with wheatbran, wheatbran and sawdust, coffee grounds, chicken manure, or mixture of different composts with and without 5% (w/w) crab shell powder either once (5%, w/w) or continuously (2.5%) into the test soils infested with the pathogen. In the container trials, the soil amended with composts became suppressive to disease development on the second and third cropping. The suppressive effect was notable in the soil amended with the mixture of compost with and without crab shell powder. The coffee compost lowered soil pH but became suppressive to the disease after modifying the soil pH. In the field trial using the mixture of the different composts containing 5% crab shell powder, a combination of 5% before the first cropping and 2.5% every second cropping gave stable disease control and promoted plant growth. After compost amendment, populations of fungi, bacteria and actinomycetes as measured by dilution plate counting and the total microbial activity as evaluated by fluorescein diacetate hydrolysis increased and population of the pathogen gradually decreased. These phenomena were especially notable in soils amended with the mixture of different composts. These results indicate that diversity in the organic materials promotes higher microbial activity and population in the soil thereby enhancing disease suppressiveness.     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.