Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Transformation of Tobacco with Genes Encoding Helianthus Tuberosus Agglutinin (HTA) Confers Resistance to Peach-Potato Aphid (Myzus persicae)

The effects of the hta gene encoding Helianthus tuberosus agglutinin (HTA) on an insect in the order Homoptera were investigated. Homologous cDNAs of hta-a, hta-b, hta-c and hta-d with CaMV35S as promoter were introduced into tobacco via Agrobacterium tumefaciens. Southern blot results showed that the exogenous hta gene was inserted into the genome of host plants, and northern blot analysis confirmed that hta was expressed in transgenic plants. A bioassay with peach-potato aphid (Myzus persicae) demonstrated that transgenic plants had deleterious effects on the insect. The average population of aphids fed on transgenic T₀ plants during an 11-day assay decreased by 70%, compared controls. In transgenic plants of T₁ generation, aphid fecundity inhibitions were 53.0%(hta-b) and 64.6% (hta-c), respectively. The development of aphids was notably retarded. We conclude that hta could be a novel and promising candidate for plant transgenic engineering against homopteran insect pests.     

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T₀ and T₁generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.     

Production of stable transgenic sunflowers (Helianthus annuus L.) from wounded immature embryos by particle bombardment and co-cultivation with Agrobacterium tumefaciens

Split embryonic axes of 21-day old immature sunflower (Helianthus annuus L.) embryos were bombarded by microparticles and then co-cultured with disarmed Agrobacterium tumefaciens strain EHA105 bearing a binary vector carrying nptII and uidA genes. Apical shoot bud development and organogenesis induced on the explants led T₀ transgenic plants. Southern blot analysis revealed complex integration patterns in T₀ plants. The uidA gene segregated as a dominant trait and single-insertion events were observed in T₁ plants. Patterns similar to those of T₁ plants were observed in T₂ progeny.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpin<Subscript>Xooc</Subscript>-encoding <Emphasis Type="Italic">hrf2</Emphasis> Gene

hrf2 gene is a member of the harpin-encoding gene family of rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola. In our previous studies, we observed that harpin_Xooc could elicit hypersensitive cell death in non-host plants, induce disease and insect resistance in plants, and enhance plant growth. In this study, the rapeseed cultivar, Yangyou 4, was genetically engineered via Agrobacterium-mediated transformation to express the hrf2 gene. Polymerase chain reaction (PCR) and southern blot analyses of T₁ generation of transgenic rapeseed revealed stable integration and expression of the inserted gene hrf2. In addition, the resistance to Sclerotinia sclerotiorum was greatly enhanced. A comparison between agronomic characters of transgenic and control lines displayed significant differences in terms of plant height, stem width, number of pods per plant, number of seeds per pod, 1,000-seed weight, and seed yield per plant. Among lines with resistance to S. sclerotiorum, T₁1 had improved agronomic traits compared with controls with a 22.7% seed yield increase. These results suggest that the introduction of the hrf2 gene into rapeseed can be an effective strategy for enhancing resistance to S. sclerotiorum.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Defense-related gene expression and enzyme activities in transgenic cotton plants expressing an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> in response to interaction with <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

There are many reports on obtaining disease-resistance trait in plants by overexpressing genes from diverse organisms that encode chitinolytic enzymes. Current study represents an attempt to dissect the mechanism underlying the resistance to Rhizoctonia solani in cotton plants expressing an endochitinase gene from Trichoderma virens. Several assays were developed that provided a powerful demonstration of the disease protection obtained in the transgenic cotton plants. Transgene-dependent endochitinase activity was confirmed in various tissues and in the medium surrounding the roots of transformants. Biochemical and molecular analyses conducted on the transgenic plants showed rapid/greater induction of ROS, expression of several defense-related genes, and activation of some PR enzymes and the terpenoid pathway. Interestingly, even in the absence of a challenge from the pathogen, the basal activities of some of the defense-related genes and enzymes were higher in the endochitinase-expressing cotton plants. This elevated defensive state of the transformants may act synergistically with the potent, transgene-encoded endochitinase activity to confer a strong resistance to R. solani infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of cruciferous phytoanticipins with plant fungal pathogens: indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are

Glucosinolates represent a large group of plant natural products long known for diverse and fascinating physiological functions and activities. Despite the relevance and huge interest on the roles of indole glucosinolates in plant defense, little is known about their direct interaction with microbial plant pathogens. Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrassicin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-derivatives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arvelexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is, desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selective. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosinolates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R. solani to the less antifungal compounds indole-3-carboxaldehyde and indole-3-carboxylic acid. Diindolyl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solution and displayed low antifungal activity; neither compound appeared to be metabolized by any of the three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani or S. sclerotiorum.