Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinerea and strawberry

Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for beta-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated while no change in expression of two endochitinases was measured. In strawberry leaves, the chitinase genes were upregulated 2-12-fold, except one of the endochitinases, whereas no change in expression of the two endoglucanases was measured. The results suggest that three out of four chitinase genes of IK726 are involved in biocontrol on leaves. This is the first example of monitoring of expression of chitinolytic genes in interactions between biocontrol agents and pathogens in plant material.     

Enhanced Resistance to Botrytis cinerea Mediated by the Transgenic Expression of the Chitinase Gene ch5B in Strawberry

Plants of strawberry (cultivar Pájaro) were transformed with three defense related genes: ch5B, gln2 and ap24 using Agrobacterium tumefaciens. The ch5B gene encodes for a chitinase from Phaseolus vulgaris, while gln2 and ap24 encode for a glucanase and a thaumatin-like protein, respectively, both from Nicotiana tabacum. Sixteen transgenic lines expressing one or a combination of two defense genes were obtained. Phytopathological tests showed that two transgenic lines expressing only the ch5B gene displayed high levels of resistance to gray mold disease (Botrytis cinerea). The resistance was correlated with the presence of the foreign protein CH5B and the increase of chitinolytic activity in leaves. However, resistance toward Colletotrichum acutatum, the etiological agent of the anthracnose disease, was not enhanced in the transgenic plants. These results suggest that the ch5B gene can be used to introduce transgene-mediated resistance to gray mold in strawberry, due to the lack of natural resistance to this disease in the crop.     

Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.     

芳樟醇对灰葡萄孢生长的影响及对番茄灰霉病的防控效果

通过平板抑菌试验和孢子萌发试验研究了芳樟醇对灰葡萄孢的生长抑制作用,并通过盆栽试验进一步验证了芳樟醇对番茄灰霉病的防控效果。结果表明: 芳樟醇能够显著抑制灰葡萄孢菌丝的生长,半最大效应浓度(EC₅₀)值为0.581 mL·L^-1。孢子萌发试验中,芳樟醇能够有效抑制灰葡萄孢孢子的萌发,并表现出浓度依赖性。芳樟醇处理提高了灰葡萄孢菌菌丝体的相对电导率和丙二醛(MDA)含量,说明芳樟醇可引起氧化损伤效应导致灰葡萄孢菌的膜系统被破坏;芳樟醇处理后灰葡萄孢菌中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性较对照组分别下降了27.4%、68.9%和26.0%,说明芳樟醇抑制了灰葡萄孢菌体内的抗氧化系统。盆栽试验结果显示,芳樟醇处理的病斑直径较对照组显著降低;番茄叶片中的SOD、CAT、POD、多酚氧化酶和苯丙氨酸解氨酶活性均显著高于对照组;而MDA含量下降了41.5%,说明芳樟醇可减轻灰葡萄孢菌对番茄植株造成的氧化损伤以提高植物抗病性。综上,芳樟醇对灰葡萄孢的生长有显著抑制作用并对番茄灰霉病有较好的防治效果,研究结果可为开发新型植物源抑菌剂防控番茄灰霉病提供理论依据。     

Biological control by Clonostachys rosea as a key component in the integrated management of strawberry gray mold

Gray mold, caused by Botrytis cinerea, is an important strawberry disease. As gray mold control is difficult, there is a need to evaluate integrated methods to successfully manage the disease. The efficiency of integrating Clonostachys rosea sprays, fungicide sprays, and crop debris removal to manage gray mold was evaluated in field experiments conducted in 2006 and 2007. Leaf colonization by C. rosea (LAC), average number of B. cinerea conidiophores (ANC), gray mold incidence in both flowers (Iflower) and fruits (Ifruit), and yield were evaluated weekly. In both years, LAC was higher in the treatments with no fungicide. When compared to the check, ANC, Iflower and Ifruit were most reduced in treatments that included C. rosea sprays. Maximal reductions were achieved with the combination of C. rosea sprays, fungicide sprays and debris removal (96.62%, 86.54% and 65.33% reductions of ANC, Iflower and Ifruit, respectively). Otherwise, maximal yield (103.14% increase as compared to the check) was achieved with the combination of the three treatments. With just C. rosea sprays, ANC, Iflower and Ifruit were reduced by 92.01%, 68.48% and 65.33%, respectively, whereas yield was increased by 75.15%. Considering the individual effects, application of C. rosea was the most efficient treatment. Chemical control was effective only in plots without debris removal. Elimination of crop debris was the least effective method in reducing gray mold incidence in both flowers and fruits. The integrated control approach enhanced the efficacy of the individual methods of gray mold control and provided high strawberry yield. An important component of this integrated approach it the biological control with C. rosea.     

A new post-harvest fungicide to control fruit rot on apple and pear

PHILABUSTER is a new post-harvest fungicide developed by Janssen Pharmaceutica N.V.. It provides an advanced mould control by post-harvest treatments of citrus and pome fruit. The product is formulated as a stable suspension concentrate intended for dilution in water before use. PHILABUSTER 400 SC contains 200 g/L imazalil and 200 g/L pyrimethanil. Both active ingredients have a different single site mode of action. Imazalil inhibits ergosterol biosynthesis (DMI), whereas pyrimethanil interferes with fungal enzyme secretion and methionine biosynthesis. Due to the combination of these low risk fungicides a good anti-resistance management can be obtained. In case of existing reduced sensitivity of a population to DMI or MBC fungicides, no cross-resistance with pyrimethanil was observed. PHILABUSTER showed good activity by post-harvest treatment against key pathogens on apple and pear Penicillium expansum (blue mold), Botrytis cinerea (gray mold) and Gloeosporium spp. (lenticel rot) in small and large scale experiments with artificial or natural infections. By dip treatment of large volumes of fruit (up to 50 tons) the depletion of both active ingredients in the treatment water was low, both when plastic or wooden bins were used. Lower dose rates resulted in an inferior and inconsistent residue level of both active ingredients on fruit. Possible advantages of post-harvest treatments versus field treatments for the control of storage diseases are discussed.     

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.     

Bacillus methylotrophicus M4-96 Stimulates the Growth of Strawberry (Fragaria × ananassa ‘Aromas’) Plants In Vitro and Slows Botrytis cinerea Infection by Two Different Methods of Interaction

Bacillus methylotrophicus M4-96 is a beneficial rhizobacterium that has been isolated from the rhizosphere of maize (Zea mays). In this study, we investigated its efficacy as a plant growth promoter for strawberry in vitro, as well as its ability to induce callose deposition in leaves to reduce the severity of Botrytis cinerea infection. Two methods of plant-bacterial interaction were used: inoculation near the root and emission of volatile compounds with no physical contact. Plant biomass increased under both treatments, but with developmental parameters of the plants differentially stimulated by each method. Root inoculation increased petiole number and root length, whereas bacterial volatiles increased petiole length and root number. A chemical analysis of the bacterial culture revealed the presence of indole acetic acid (0.21 μg L⁻¹) and gibberellic acid (6.16 μg L⁻¹). Acetoin was previously identified as the major volatile produced by the bacteria, and its application to strawberry explants increased their growth and development. Furthermore, when acetoin and both phytoregulators were added to the culture media, these positive effects were enhanced. The inoculation method also affected the size and quantity of callose deposits in the leaves. Treatment with volatiles increased callose deposition in the leaves by up to five-fold, which promoted a rapid defense reaction that inhibited the incidence of gray mold by reinforcing cell wall. Taken together, our results show that B. methylotrophicus M4-96 promotes growth and induces systemic resistance in strawberry plants.

     

Screening of wild accessions resistant to gray mold (Botrytis cinerea Pers.) in Lycopersicon

Tomato gray mold (Botrytis cinerea Pers.) is a common disease worldwide, and often causes serious production loss by infecting leaves, stems, flowers and fruits. Presently, no resistant cultivars are available. To find new breeding materials for gray mold resistance, assessment for resistance of the leaflet and stem in six tomato cultivars, 44 wild tomato accessions and a Solanum lycopersicoides accession was performed. Although no correlation was observed (r=−0.127^ns) between resistance of the leaflet and the stem, L. peruvianum LA2745, L. hirsutum LA2314 and L. pimpinellifolium LA1246 showed high resistance both in the leaflet and in the stem. Particularly, in the leaves of LA2745, no lesions were observed even more than two weeks after the inoculation with conidia, and F1s between a cultivated tomato and LA2745 also showed high resistance as observed in LA2745. From these results, LA2745 is thought to be a promising material for breeding gray-mold resistant cultivars.     

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

The potential of the active ingredient of a commercial bioinsecticide, XenTari^® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari^® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.     

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.     

番茄内生菌St24的鉴定及其对灰霉病的生防作用

从番茄植株根茎部分离到1株有抑菌活性的植物内生放线菌菌株St24,对其分类地位以及对灰霉病菌的防治效果进行研究.结果表明: 菌株St24为酒红链霉菌.St24发酵液经石油醚萃取得到的粗提物对多种病原菌有抑制作用,其中对灰霉病菌的抑制作用最强,抑制菌丝生长的EC₅₀为11.78 mg·L^-1.粗提物处理灰霉菌后,菌丝量减少,菌丝体皱缩、断裂、原生质外渗,菌体细胞表面有许多瘤状畸形.处理后的灰霉病菌培养液在260 nm处比对照多出现一吸收峰,说明粗提物对病原菌的细胞膜透性有影响.经盆栽试验, St24发酵液对番茄灰霉病有保护和治疗的作用,100 mg·L^-1粗提物叶面喷雾的保护作用效果最好,24 h后防效达到94.3%,120 h后为85.4%.     

Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea

In the gray mold fungus Botrytis cinerea, spore germination and plant infection are stimulated in the presence of nutrients, in particular sugars. Applied at micromolar concentrations, fructose is a more potent inducer of germination than glucose. To test whether preferred fructose uptake is responsible for this effect, and to study the mechanism of fructose transport in B. cinerea, a gene (frt1) encoding a fructose transporter was cloned. FRT1 is highly similar to recently identified fructose transporters of yeasts, but much less to other fungal hexose transporters characterized so far. By using a hexose uptake deficient yeast strain for expression, FRT1 was found to be a high affinity proton coupled symporter specific for fructose but not for glucose. B. cinerea frt1 disruption mutants were created and showed normal vegetative growth and plant infection, but a delay in fructose-induced germination when compared to wild-type. Sugar uptake experiments with both wild-type and mutant conidia showed a higher affinity for glucose than for fructose. Thus, we propose that the specific effect of fructose on germination is not due to transport but rather to an as yet unknown intracellular sensing.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell-free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B. cinerea. An assays based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.     

Release of lipoxygenase products and monoterpenes by tomato plants as an indicator of Botrytis cinerea-induced stress

Changes in emission of volatile organic compounds (VOCs) from tomato induced by the fungus Botrytis cinerea were studied in plants inoculated by spraying with suspensions containing B. cinerea spores. VOC emissions were analysed using on-line gas chromatography–mass spectrometry, with a time resolution of about 1 h, for up to 2 days after spraying. Four phases were delimited according to the starting point and the applied day/night rhythm of the experiments. These phases were used to demonstrate changes in VOC flux caused by B. cinerea infestation. Tomato plants inoculated with B. cinerea emitted a different number and amount of VOCs after inoculation compared to control plants that had been sprayed with a suspension without B. cinerea spores. The changes in emissions were dependent on time after inoculation as well as on the severity of infection. The predominant VOCs emitted after inoculation were volatile products from the lipoxygenase pathway (LOX products). The increased emission of LOX products proved to be a strong indicator of a stress response, indicating that VOC emissions can be used to detect plant stress at an early stage. Besides emission of LOX products, there were also increases in monoterpene emissions. However, neither increased emission of LOX products nor of monoterpenes is specific for B. cinerea attack. The emission of LOX products is also induced by other stresses, and increased emission of monoterpenes seems to be the result of mechanical damage induced by secondary stress impacts on leaves.     

Characterization of new bacterial biocontrol agents Acinetobacter,Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea

A collection of 282 bacterial isolates from the rhizosphere and different organs of healthy field-grown grapevine plants was obtained and screened for their ability to protect grapevine leaves against Botrytis cinerea, the causal agent of gray mold. Twenty-six strains effectively controlled B. cinerea infections on leaves. After phenotypic and molecular analysis, seven strains were identified as Pseudomonas fluorescens PTA-268 and PTA-CT2, Bacillus subtilis PTA-271, Pantoea agglomerans PTA-AF1 and PTA-AF2, and Acinetobacter lwoffii PTA-113 and PTA-152. In vitro antifungal experiments showed that from these seven strains, only PTA-AF1 and PTA-CT2 exhibited a direct antagonism against B. cinerea. Furthermore, the biocontrol activity of the seven bacteria was associated with differential induction of defense-related responses lipoxygenase, phenylalanine ammonia-lyase and chitinase in grapevine leaves. Our results show that the selected bacteria can efficiently protect grapevine leaves against gray mold disease through an induction of plant resistance and in some cases by an additional antagonistic activity.     

Cloning,expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler)

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1). Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2). Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry. RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The Fxacad1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.