Spore Germination Fluid Research Articles

Alternaria alternata causing black spot of peach produces a host-specific toxin

Here we report a new host-specific toxin that is produced by Alternaria alternata and is the eight reported for the fungus. The causal pathogen of black spot of peach produced a toxin in culture filtrates and in spore germination fluids. Toxin productivity during spore germination was closely related to pathogenicity of the pathogen. The degree of sensitivity of host plants to the toxin was consistent with that of susceptibility to the pathogen. Further, the toxin enabled infection by conidia of saprophytic A. alternata in peach leaves. Several cultivars showed resistance to the toxin and the pathogen, although no cultivars had an immune response. The toxin fit the criteria of a host-specific toxin and was designated AP-toxin.

Elicitor(s) production is involved in red-light-induced resistance during spore germination of Bipolaris oryzae in the presence of host tissues

In the present study, the effect of red light on the infection behavior of Bipolaris oryzae on rice leaves and the effects of chemical inhibitors and spore germination fluid (SGF) of B. oryzae on red-light-induced resistance against brown spot disease were investigated. Red light irradiation and natural light did not differ significantly with respect to their effect on spore germination and appressorium formation of B. oryzae 24 h after inoculation. However, formation of infection hyphae was significantly inhibited under red light compared to that under natural light. Pretreatment with the photosynthetic inhibitor 3-(3,4-dichorophenyl)-1,1-dimethylurea (DCMU) or with the aromatic amino acid inhibitor N-(phosphonomethyl) glycine (glyphosate) for 24 h inhibited the development of resistance in rice leaves. To elucidate the elicitor(s) produced during the B. oryzae–rice plant interaction, the effect of SGF prepared in the absence (A-SGF) or presence (P-SGF) of rice leaves on red-light-induced resistance was investigated. When rice plants were pretreated with A-SGF or P-SGF for 24 h, P-SGF had elicitor activity under red light, but not under natural light or with A-SGF. These results suggest that germinating spore of B. oryzae produced an elicitor(s) under red light conditions in a host-dependent manner. In conclusion, we hypothesize that an unidentified elicitor(s) produced at an early stage of fungal infection during the B. oryzae–rice interaction contributes to the inhibition of cell death in rice leaves under red light and enhances resistance-related tryptophan and phenylpropanoid pathways.

The sensitivity of Canadian wheat genotypes to the necrotrophic effectors produced by Pyrenophora tritici-repentis

Pyrenophora tritici-repentis (Ptr), the cause of tan spot of wheat, is a foliar pathogen of economic importance worldwide. Tan spot development is associated with the production of necrotrophic effectors (Ptr ToxA, Ptr ToxB and Ptr ToxC) that serve as fungal pathogenicity and/or virulence factors. The objective of this study was to investigate the sensitivity to each of these effectors in a collection of 100 Canadian wheat cultivars representing all wheat classes and developed over a century by various Canadian breeding programmes. Heterologously expressed, His-tagged Ptr ToxA and Ptr ToxB, and spore germination fluids possessing putative Ptr ToxC activity, were produced and used to screen the cultivars for sensitivity. Of the 100 wheat cultivars tested, 92 were sensitive to at least one effector, 68 were sensitive to Ptr ToxA, 63 were sensitive to Ptr ToxC and 24 were sensitive to Ptr ToxB. Only eight cultivars were insensitive to all three effectors. These results reflect the widespread sensitivity in Canadian wheat cultivars to these necrotrophic effectors, with sensitivity to Ptr ToxA and the putative Ptr ToxC found consistently to be more common than Ptr ToxB sensitivity. The predominance of Ptr ToxA and Ptr ToxC sensitivity in Canadian wheat is consistent with the prevalence of races 1 (Ptr ToxA + Ptr ToxC producer) and 2 (Ptr ToxA producer) of Ptr in North America, and could reflect a competitive advantage for Ptr ToxA-producing isolates in the pathogen population.

Phakopsora pachyrhizi induces defense marker genes to necrotrophs in Arabidopsis thaliana

Phakopsora pachyrhizi is a rust fungus that causes Asian soybean rust disease. In the nonhost plant Arabidopsis thaliana penetrated epidermis cells initiate cell death associated with mRNA transcript accumulation of PDF1.2, a marker gene for defense against necrotrophic pathogens. PDF1.2 activation occurred after both inoculation of A. thaliana leaves with P. pachyrhizi spores and upon their treatment with spore germination fluid, suggesting PDF1.2 activation by secreted fungal effectors. These are likely (glyco)proteins. Together, P. pachyrhizi seems to feign a necrotroph pathogen during early stages of infection to circumvent plant defense to biotrophs.

Release of elicitors from rice blast spores under the action of reactive oxygen species

Effects of reactive oxygen species (ROS) on the release of putative elicitors from spores of rice blast causal fungus Magnaporthe grisea (Hebert) Barr were studied. While studying the influence of exogenous ROS, the spores were germinated for 5 h in the presence of 50 μM H2O2 and then treated with catalase to decompose hydrogen peroxide. The spore germination fluid was then boiled to inactivate catalase. When the resulting diffusate was applied onto rice (Oryza sativa L.) leaves, it caused necroses and stimulated superoxide (O2−) production. Both effects were observed with the resistant rice cultivar but not with the cultivar susceptible to the fungal strain. The susceptible cultivar did not acquire resistance to challenge with fungal spores, which were applied one day after the treatment. The fractionation of the spore diffusate showed that both low- and high-molecular compounds (mol wt 3 kD, respectively) should be present in combination to induce O2− production by leaves. The diffusates from spores germinated in water also caused necroses and stimulated O2− generation, though to a weaker extent than diffusates from spores germinated in H2O2. The effect of diffusates from spores germinated in water was abolished by catalase or superoxide dismutase added initially to the spore suspension. The results suggest that germinating spores of M. grisea are able to release elicitors and this ability depends on ROS formation by spores. Presumably, the yield of elicitors is increased additionally if fungus M. grisea is stressed or subjected to exogenous ROS. The described phenomena may be involved in incompatibility mechanisms.

Alternaria brassicae Produces a Host-Specific Protein Toxin from Germinating Spores on Host Leaves

Spore suspensions of Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, were incubated on the leaves of cabbage (B. oleracea) and spore germination fluid (SGF) was collected after 48 h. A high molecular weight (HMW) fraction (>10 kDa) was separated from the SGF by ultrafiltration. In a detached leaf assay, the HMW fraction induced visible symptoms only on host leaves and the toxicity was lost by treatment with proteinase K or heat at 60 degrees C for 15 min, indicating the presence of host-specific protein toxin(s). A protein toxin in the HMW fraction was purified by several chromatography steps. The toxin induced water-soaked symptoms followed by chlorosis at concentrations of 0.5 to 1 microg/ml on host leaves, but not on nonhost leaves even at 50 microg/ml. The toxin also had infection-inducing activity when added to spore suspension of a nonpathogenic isolate of A. alternata, causing symptoms similar to the infection of A. brassicae only on host leaves. These results indicate that a new host-specific protein toxin named ABR-toxin is released from germinating spores of A. brassicae on host leaves. ABR-toxin migrated as a protein of 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of ABR-toxin was estimated to be approximately 7.0 and 21 N-terminal amino acid residues were sequenced.

Destruxin B produced by Alternaria brassicae does not induce accessibility of host plants to fungal invasion

It has been reported that Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, produces a host-specific or host-selective toxin (HSTs) identified as destruxin B. In this study, the role of destruxin B in infection of the pathogen was investigated. Destruxin B purified from culture filtrates (CFs) of A. brassicae induced chlorosis on host leaves at 50–100 μg ml −1, and chlorosis or necrosis on non-host leaves at 250–500 μg ml −1. Destruxin B was detected in spore germination fluids (SGFs) on host and non-host leaves, but not in a sufficient amount to exert toxicity to host plants. When spores of non-pathogenic A. alternata were combined with destruxin B at 100 μg ml −1 and inoculated on the leaves, destruxin B did not affect the infection behavior of the spores. Interestingly, SGF on host leaves allowed non-pathogenic spores to colonize host leaves. Moreover, a high molecular weight fraction (>5 kDa) without destruxin B obtained by ultrafiltration of SGF had host-specific toxin activity and infection-inducing activity. From these results, we conclude that destruxin B is not a HST and does not induce the accessibility of the host plant which is essential for colonization of the pathogen. In addition, the results with SGF imply that a high molecular weight HST(s) is involved in the host–pathogen interaction.

Quantitative analysis of secondary metabolites produced by Chaetomium globosum Krunze ex Fr.

Chaetomium globosum Krunze ex Fr. (Cg) has been identified as a potential antagonist of Bipolaris sorokiniana and Ascochyta rabei . This biocontrol agent has been found to be producing antifungal metabolites. Six different isolates of Cg were characterized for the production of antifungal metabolites. The quantitative estimation of crude extracts showed maximum production by isolate Cg3 (47.2μg/mg mycelium) followed by Cg2 (44.2μg/mg). Isolate Cgl produced minimum quantity of metabolites (7.44μg/mg). Crude extracts of secondary metabolites from all the isolates of C. globosum when resolved by thin layer chromatography showed that the isolate of Cgl produced only two compounds of R r value 0.17 and 0.48, while isolate Cg2 produced thirteen compounds, followed by 11 compounds produced by isolate Cg3, The metabolite from spore germination fluid of isolate Cg2, when resolved on TLC plate along with crude extracts from culture filtrate of Cg2, showed production of about same number of compounds. One of the purified compounds from isolate Cg2 when used for bioassay against Bipolaris sorokiniana, Rhizoctonia solani, Fusarium udum and Macrophomina phaseolina pathogens proved effective in inhibiting the growth up to 70 percent.

Detoxification of α-tomatine by tomato pathogens Alternaria alternata tomato pathotype and Corynespora cassiicola and its role in infection

In tomato plants, α-tomatine, a steroidal glycoalkaloid saponin, inhibits fungal growth. Tomato pathogens that produce host-specific toxins, Alternaria alternata tomato pathotype causing Alternaria stem canker and Corynespora cassiicola causing Corynespora target spot, were investigated for sensitivity to α-tomatine. Although spore germination of A. alternata pathogenic and nonpathogenic to tomato and of C. cassiicola pathogenic to tomato was not affected by 0.1 mM α-tomatine, spore germination of C. cassiicola nonpathogenic to tomato was significantly inhibited. This result showed that A. alternata, regardless of its pathogenicity, and only the C. cassiicola pathogenic to tomato are resistant to α-tomatine. Germinating spores of A. alternata and C. cassiicola resistant to α-tomatine detoxified α-tomatine by degrading it to a less polar product. After inoculation of tomato leaves, spores of A. alternata and C. cassiicola nonpathogenic to tomato germinated and formed appressoria, but did not form infection hyphae in host tissues. When a host-specific toxin (CCT-toxin) produced by C. cassiicola pathogenic to tomato was added to nonpathogenic spores, colonization within leaves was observed in A. alternata, but not in C. cassiicola. On the other hand, when spores of C. cassiicola nonpathogenic to tomato were suspended in spore germination fluid of nonpathogenic A. alternata with α-tomatine detoxification activity, the fungus could be induced to colonize leaves in the presence of CCT-toxin. These results indicate that A. alternata tomato pathotype and C. cassiicola pathogenic to tomato detoxify α-tomatine during infection and that this detoxification is essential for host colonization by pathogens that produce host-specific toxins.

Pyrichalasin H production and pathogenicity of Digitaria‐specific isolates of Pyricularia grisea

SUMMARY Culture filtrates from 72 isolates of Pyricularia, grouped into 13 rDNA types, were analysed via HPLC. Of these isolates, 31 (r9 DNA type) from crabgrass (Digitaria sanguinalis), one (r9 DNA type) from pangolagrass (Digitaria smutsii) and six (r8 DNA type) from Digitaria horizontalis produced 20-280 microg pyrichalasin H per millilitre of culture. These same isolates were pathogenic on five Digitaria species. Interestingly, two isolates, KM-1 and Br 29, which were originally isolated from Digitaria plants, did not produce pyrichalasin H, nor caused blast lesion on Digitaria plants. Because these two isolates were identified as Digitaria pathogens by PCR analysis using Digitaria-specific primers, they are likely to be mutants lacking pyrichalasin H production. Isolates that belonged to the remaining 11 rDNA types did not produce pyrichalasin H and were avirulent to Digitaria plants. Therefore, the virulence of Pyricularia on Digitaria plants correlates with pyrichalasin H production. Pyrichalasin H was also present in spore germination fluid of a crabgrass isolate (IBDS 5-1-1), but not in that of isolates from rice, foxtail millet, finger millet, common millet and wheat. In addition, pyrichalasin H was detected in host leaves infected with IBDS 5-1-1, but not in leaves from other plants infected with compatible Pyricularia isolates. Pretreatment of leaf sheaths of crabgrass with 3 microg/mL pyrichalasin H led to the penetration and colonization by non-host isolates. Overall, these results indicate that production of pyrichalasin H is responsible for the genus-specific pathogenicity of Digitaria isolates.

Increased tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea.

Sekiguchi lesion (sl)-mutant rice infected with Magnaporthe grisea showed increased light-dependent tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) activities. TDC and MAO activities were observed before the penetration of M. grisea to rice cells and maintained high levels even after Sekiguchi lesion formation. Light-dependent expression of TDC gene was observed in leaves inoculated with M. grisea before Sekiguchi lesion formation. Spore germination fluid (SGF) of M. grisea also induced Sekiguchi lesion formation accompanied by increased enzymes activities and tryptamine accumulation. Sekiguchi lesion was also induced by treatments with tryptamine and beta-phenylethylamine, which are substrates for MAO, but was not induced by non-substrates such as indole-3-propionic acid, (+/-)-phenylethylamine and tryptophan under light. Light-dependent induction of Sekiguchi lesion by tryptamine was significantly inhibited in the presence of MAO inhibitors, metalaxyl and semicarbazide, and H2O2-scavengers, ascorbic acid and catalase. H2O2 in M. grisea-infected leaves with and without Sekiguchi lesions was demonstrated directly in situ by strong 3,3'-diaminobenzidine (DAB) staining. On the other hand, H2O2 induced Sekiguchi lesions on leaves of cv. Sekiguchi-asahi under light, but not in darkness. This difference was associated with the decrease of catalase activity in infected leaves under light and the absence of decrease in darkness. We hypothesize that the H2O2-induced breakdown of cellular organelles such as chloroplasts and mitochondria in mesophyll cells may cause high TDC and MAO activities and the development of Sekiguchi lesion, and that the sl gene products in wild-type rice may function as a suppressor of organelle breakdown caused by chemical or environmental stress.

Purification of a Necrosis-Inducing, Host-Specific Protein Toxin from Spore Germination Fluid of Alternaria panax

ABSTRACT Spore germination fluid of Alternaria panax, the causal agent of Alternaria blight of American ginseng (Panax quinquefolius), collected from water droplets or aqueous ginseng leaf extracts produced visible water-soaked lesions on wounded, detached leaflets after incubation for 48 h. Maximum development of brown, necrotic spots occurred 4 to 5 days after inoculation on attached and detached ginseng leaflets. Of 15 plant species tested, only American ginseng was susceptible to applications of spore inoculum or spore germination fluid. The phytotoxic activity of the spore germination fluid was destroyed by heat and treatment with proteinase A. The phytotoxic factor was retained by an ultrafiltration membrane with a 30-kDa molecular mass cut-off. Purification of the phytotoxic protein, named AP-toxin, was performed by anion exchange and gel filtration chromatography. Bioactive fractions eluted as a single peak. By comparison with protein standards, a molecular mass of 35 kDa was estimated for the native protein. The denatured protein toxin also had a mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Production of the protein toxin was induced on American ginseng leaflets and water extracts of ginseng leaves but not on leaves of other nonhost plants and their water extracts. The results show that A. panax produces a host-specific, proteinaceous toxin during colonization and pathogenesis of ginseng leaves.

Red‐Light‐Induced Resistance in Broad Bean (Vicia faba L.) to Leaf Spot Disease Caused by Alternaria tenuissima

AbstractThe effect of different light wavelengths on the development of lesions induced by Alternaria tenuissima in broad bean leaves was investigated. Lesion development was completely suppressed in red‐light‐irradiated broad bean leaflets, irrespective of isolate or spore concentration. Pre‐treatment of leaflets with red light for 24 h before inoculation also suppressed lesion development. Alternaria tenuissima failed to produce infection hyphae in red‐light‐irradiated broad bean leaflets. These results indicate that disease suppression in broad bean leaflets is due to light‐induced resistance. Spore germination fluid (SGF) of A. tenuissima allowed non‐pathogenic Alternaria alternata to infect wounded and unwounded broad bean leaflets kept in the dark, results suggesting that SGF induced susceptibility. Red light suppressed susceptibility induced by A. tenuissima SGF; thus, lesion formation and development were suppressed when leaflets inoculated with the spores of A. alternata suspended in A. tenuissima SGF were kept under red light. From these results, we conclude that red light induced resistance in broad bean to leaf spot disease caused by A. tenuissima, and that SGF induced susceptibility of broad bean leaflets to a non‐pathogenic isolate of A. alternata.

Host Species-Specific Protoplast Damaging Activity of Spore Germination Fluids of Blast Pathogen Magnaporthe grisea

The effect of spore germination fluids (SGFs) of rice and ragi (Eleusine coracana) limited forms of Magnaporthe grisea was studied on the protoplasts of host and non-host species to determine their host specificity. The concentrated SGFs of rice strain of M. grisea exhibited strong disruptive activity against the protoplasts of two rice cultivars, Fukunishiki and Caloro, but had little effect on the protoplasts of ragi cv. VL 17 and Podophyllum hexandrum. However, the SGFs of ragi strain of M. grisea showed a high tendency to disrupt ragi protoplasts but inflicted less damage on protoplasts of the two rice cultivars and P. hexandrum. These results suggest that SGFs of M. grisea contain some factor(s) which exhibit host species-specific activity and may be the determinants of basic compatibility.

Induction of defense responses by synthetic glycopeptides that have a partial structure of the elicitor in the spore germination fluid of Mycosphaerella pinodes.

A high molecular weight elicitor (> 70 kDa) from spore germination fluid of a pea pathogen, Mycosphaerella pinodes, has a partial structure of beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man, which is O-glycosidically attached to serine in the protein moiety. To elucidate the minimum structure for the elicitor activity to pea plants, the effects of nine glycopeptides including beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man-O-Ser (No. 1) to [beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man]3-O-Ser3-Pro3 (No. 9) on the infection by M. pinodes, superoxide generation and ATPase activity were measured. The glycopeptides [beta-D-Glc-(1-->6)-alpha-D-Man-(1-->6)-D-Man]-O-Ser2-Pro2 (No. 3) to No. 9 induced rejection reaction of pea tissue against M. pinodes. The glycopeptides No. 3 to No. 9 also induced superoxide generation on uninjured pea leaves. Moreover, the glycopeptides No. 3 to No. 9 induced in vitro the activation of cell wall-bound ATPase and superoxide generation system in the protein fraction solubilized from pea cell wall. The results indicate that the synthetic glycopeptides, No. 3 to No. 9, are available to analyze the signal transduction cascade leading to defense responses and the receptor for the elicitor.

Production of a host-specific toxin by germinating spores ofAlternaria brassicicola

Spore suspensions ofAlternaria brassicicolapathogenic toBrassicaspp. were incubated on the surface ofBrassicaleaves. The spore germination fluid was collected and examined for biological activity. In a detached leaf assay, the fluid induced water-soaking symptoms followed by brown necrotic lesions onBrassicaleaves susceptible to the pathogen but did not cause visible symptoms on non-host leaves. When spores of the non-pathogenic speciesAlternaria alternatawere combined with the fluid and inoculated on the leaves, the spores could invadeBrassicaleaves, just as the pathogen did. These results indicate thatA. brassicicolaproduces a host-specific toxin(s) and releases it during spore germination on the leaves. Toxin activity of the fluid was retained by an ultrafiltration membrane with a 5 kDa cut off. The toxin was purified by ion exchange chromatography and gel filtration HPLC. The molecular weight of the toxin was estimated to be 35 kDa by SDS-polyacrylamide gel electrophoresis. The activity of the toxin was heat labile and was also lost after treatment with proteinase K. Thus, the toxin named AB-toxin is probably a protein, the first described among the many host-specific toxins produced byAlternariaspecies.

Morphogenic Regulation of Pathotoxin Synthesis inCochliobolus carbonum

The fungusCochliobolus carbonumcauses leaf spot disease of maize. Highly virulent isolates of the pathogen produce a host-selective, peptide toxin that is active against susceptible genotypes of maize. Prior to infection, spores must germinate and differentiate appressoria, structures specialized for leaf penetration. Analysis of spore germination fluids by plasma desorption mass spectrometry, which allowed detection of as little as 0.5 ng toxin, revealed that spores induced to form appressoriain vitrosynthesized and released the toxin at a time coincident with maturation of appressoria. Spores incubated under conditions that did not induce appressorium formation failed to produce toxin. These observations indicate that synthesis of the host-selective toxin, which is essential for successful pathogenesis of maize byC. carbonum,is regulated by infection-related morphogenesis.

Phytotoxicity of solanapyrones produced by the fungus Ascochyta rabiei and their possible role in blight of chickpea ( Cicer arietinum)

Solanapyrones A, B and C have been extracted from the culture filtrates and spore germination fluids of Ascochyta rabiei and purified by reverse phase HPLC. Maximum production of solanapyrones was observed in the chickpea seed extract medium at the onset of sporulation. Supplementation of the culture medium with the extract of the host plant stimulated solanapyrone production. Production of solanapyrones by different isolates of Ascochyta rabiei was well correlated with the degree of pathogenicity of these isolates on chickpea. Phytotoxicity of solanapyrones to different chickpea varieties in a host-selective and concentration-dependent manner suggests their role as a factor in the development of chickpea blight.

Evidence of a Host-Specific Chlorosis Toxin fromPyrenophora tritici-repentis,the Causal Agent of Tan Spot of Wheat

Spore germination fluid, cell-free culture filtrate, and intercellular washing fluid from race 5 of Pyrenophora tritici-repentis contained a host-specific toxin which elicited extensive chlorosis on Katepwa and 6B662, characteristic of the chlorotic symptom induced by the fungus on the same wheat genotypes. These three sources of toxin showed equivalent host specificity. Other races (including races I and 3) which are known to induce chlorosis in different wheat genotypes than does race 5 did not produce a detectable level of a chlorosis toxin, as assessed by bioassay, when grown in a still liquid culture or during spore germination. Of the 15 plant species tested, only wheat (genotypes 6B662, Katepwa, and Neepawa) and triticale (cv. Banjo) developed distinct chlorosis when inoculated with race 5 or infiltrated with its spore germination fluid or partially purified culture filtrate. F 2 progenies from a cross between race 5-susceptible and race 5-resistant wheat genotypes were evaluated. Susceptibility of the seedlings to race 5 and their sensitivity to its toxin cosegregated. A ratio of 1 :3 (resistant/susceptible) was observed, suggesting the involvement of a single, dominant locus controlling the reaction to the fungus and the toxin. The chlorosis toxin appeared to be a pathogenicity factor and was designated as Ptr-chlorosis toxin.

Production of a Host‐Specific Toxin by Germinating Spores of Alternaria tenuissima Causing Leaf Spot of Pigeon Pea

Production of a host‐specific toxin by Alternaria tenuissima, the cause of pigeon pea leaf spot, was investigated in spore‐germination fluids (SGF). The SGF selectively induced necrosis on pigeon pea leaves in a deteched leaf assay. Necrotic lesions were observed when a toxin from SGF was applied onto detached young leaves of the pigeon pea cultivar Bahar at concentration as low as 5 ng/ml. The resistant line Tanzania and nonhosts tolerated at least 20,000 times higher concentration of the toxin. The differential activity of the toxin on hosts and nonhosts of the fungus, as well as on susceptible and resistant cultivars or lines, suggested host‐specific property of the toxin. At a concentration of 10 ng/ml, the toxin induced susceptibility of pigeon pea leaves to a non‐pathogenic isolate of Alternaria alternata. The toxin possibly plays a role as a disease determinant of A. tenuissima, because the toxin was released from germinating spores as early as 3 h of incubation andthe, amount detected within 9 h was about 6 times of the concentration required for necrotic toxicity.