Carnation Wilt Research Articles

Molecular characterization and biocontrol potential of Trichoderma spp. against Fusarium oxysporum f. sp. dianthi in carnation

BackgroundCarnation, a major cut flower product cultivated economically in Iran, faces economic challenges due to the devastating Fusarium wilt caused by Fusarium oxysporum f. sp. dianthi (Fod). To address this issue, twenty-five Trichoderma harzianum and T. viride isolates were collected from the rhizosphere soil of three Iranian provinces: Tehran, Markazi, and Fars. RAPD-PCR was applied to analyze the genetic relatedness of the isolates.ResultsThe RAPD profiles showed genetic diversity among the isolates, with two major clusters. The antagonistic potential of the twenty isolates was evaluated against the carnation wilt caused by Fod. The results showed that Th1, Th7, and Th2 isolates of T. harzianum significantly inhibited Fod mycelial growth (58, 56.5, and 48.6%, respectively). Among T. viride isolates, Tv5, Tv4, and Tv7 exhibited the highest antifungal ability to inhibit mycelial growth. All investigated isolates of Trichoderma secreted volatile compounds that hindered Fod mycelial growth, with isolates of T. harzianum ranging from 9.3 to 67.5% inhibition and those of T. viride from 25.2 to 50.2%. Additionally, the experiment on competitive saprophytic ability indicated that maximum colonization occurred with Th1, Th7, and Th2 isolates at 78.2, 70.8, and 69.8%, respectively. Lastly, the greenhouse experiment showed a complete pathogen eradication or significant inhibition in the infected carnation after T. harzianum and T. viride. Conversely, control treatment with the Fod pathogen died after 90 days.ConclusionsThe investigation suggested that Trichoderma spp. could be a potential biocontrol agent to mitigate Fusarium wilt in carnation and improve production quality, replacing chemical pesticides.

Study on Genetic Diversity of Fusarium oxysporum f.sp. dianthi Based on Vegetative Compatibility Groups and Races in Five Provinces of Iran

Fusarium wilt of carnation caused by Fusarium oxysporum f.sp. dianthi (Fod) W.C.Snyder & H.N. Hensen, is the most important fungal disease in all main carnation growing regions of Iran. In 2020, sampling of infected plants was randomly carried out from carnation greenhouses in five provinces located in the North (Gilan, Golestan, and Mazandaran) and Middle (Tehran and Markazi) of Iran. Eighty-eight fungal isolates were identified as Fod by morphological characters (based on Nelson’s key identification), molecular method (PCR amplified of ITS region), and pathogenicity tests (according to the root dipping on sensitive cv. Rendezvous). In order to investigate on biodiversity of Iranian Fod isolates based on Vegetative Compatibility Groups (VCGs) and races, 70 isolates were randomly selected. These isolates were compared with eight Fod isolates from the collection of Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali (Di.Va.P.R.A) Patologia vegetale, Italy; which were used as references for races and VCGs. For DNA amplifying, primers R 1.8, R 2.1, and R 4.2 were employed. Amplified fragments of 295 bp and 564 bp were identified to race 1 (or 8; because these primers applied are not able to discriminate between the races), and race 2 (or 5 or 6), respectively. Furthermore, two VCG 0021 and VCG 0022 were identified amongst the Iranian Fod isolates. The relationship between the races and VCGs demonstrated that all race 1 (23 isolates) were categorized in VCG 0022, and race 2 (44 isolates) were grouped in VCG 0021. In addition, three isolates did not produce any amplification.

Molecular characterization of <em>Fusarium oxysporum</em> f.sp. dianthi and evaluation of fungicides against <em>Fusarium wilt</em> of carnation under protected cultivation

Fusarium wilt, caused by Fusarium oxysporum f.sp. dianthi (FOD), is one of the most devastating carnation diseases globally, resulting in significant crop loss (40-70%). The purpose of this study was to determine the efficacy of several fungicides against Fusarium wilt of carnation in vitro and in vivo under protected cultivation. The disease infected plant samples were collected, and pathogen isolates were confirmed through morphological and molecular techniques. The rDNA sequences of isolates had 97-100% similarity with other sequences of FOD available in Genbank and the isolates were identified as Fusarium oxysporum f.sp. dianthi. Phylogenetic analysis of rDNA sequences revealed 62-99% sequence similarity among the isolates. Further, we evaluated nine different fungicides against the virulent pathogenic isolate CFODTNAU1 under in vitro and in vivo. Three different concentrations such as 500, 1000 and 1500 ppm were evaluated on mycelial growth of the pathogen under in vitro and percent inhibition over control was calculated. Among the fungicides, three of them namely, tebuconazole 50% + trifloxystrobin 25% WG, tebuconazole 25.9% SC and azoxystrobin 23% EC completely inhibited (100% inhibition) the mycelial growth of the fungus in vitro. Under protected cultivation, application of tebuconazole 50% + trifloxystrobin 25% WG through root dipping and soil drenching @ 1.0 g/L recorded 11.11% of wilt incidence compared to control (29.71% wilt incidence), which was 59.99% reduction over control. Besides, this fungicide also increased the stem length, earliness of flowering and yield than the control and other treatments. Thus, the fungicide tebuconazole 50% + trifloxystrobin 25% WG can be used for amelioration and control of wilt disease in carnation.

Mycelium Dispersion from Fusarium oxysporum f. sp. dianthi Elicits a Reduction of Wilt Severity and Influences Phenolic Profiles of Carnation (Dianthus caryophyllus L.) Roots.

The fungal pathogen Fusarium oxysporum f. sp. dianthi (Fod) is the causal agent of the vascular wilt of carnation (Dianthus caryophyllus L.) and the most prevalent pathogen in the areas where this flower is grown. For this reason, the development of new control strategies against Fod in carnation has been continuously encouraged, in particular those based on the implementation of plant resistance inducers that can trigger defensive responses to reduce the disease incidence, even at lower economical and environmental cost. In the present study, the effect of the soil supplementation of a biotic elicitor (i.e., ultrasound-assisted dispersion obtained from Fod mycelium) on disease severity and phenolic-based profiles of roots over two carnation cultivars was evaluated. Results suggest that the tested biotic elicitor, namely, eFod, substantially reduced the progress of vascular wilting in a susceptible cultivar (i.e., ‘Mizuki’) after two independent in vivo tests. The LC-MS-derived semi-quantitative levels of phenolic compounds in roots were also affected by eFod, since particular anthranilate derivatives, conjugated benzoic acids, and glycosylated flavonols were upregulated by elicitation after 144 and 240 h post eFod addition. Our findings indicate that the soil-applied eFod has an effect as a resistance inducer, promoting a disease severity reduction and accumulation of particular phenolic-like compounds.

Ecofriendly management of fusarium wilt of carnation (Dianthus caryophyllus L.) by application of organic amendments

The effect of different organic inputs viz., neem cake, mauha cake, coirpith and vermi compost were evaluated against the wilt disease incidence and the growth and yield of carnation. Preliminary screening of different oil cakes and water extracts of organic manures against the growth Fusarium oxysporum f. sp. dianthi was carried out by poisoned food technique under laboratory conditions. Among the oil cake and organic manures tested in vitro, neem cake (10%) effectively reduced the growth of Fusarium oxysporum f. sp. dianthi which recorded a minimum mycelial growth of 1.76 cm with maximum per cent inhibition of 80.44 over control. In organic field experiment, neem cake significantly reduced the disease incidence (12.47 PDI), followed by mauha cake and vermi compost which recorded 14.45 and 16.04 PDI of wilt incidence, respectively. The use of vermicompost significantly increased the growth and yield parameters viz., stalk length (85.25 cm), stalk girth (3.88 cm), no of branches (3.85/plant), no of flowers (3.11/plant) and flower diameter (4.32 cm).

Fusarium oxysporum f. sp. dianthi virus 1 Accumulation Is Correlated with Changes in Virulence and Other Phenotypic Traits of Its Fungal Host.

Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) was detected in isolate 116 (116V+) of Fusarium oxysporum f. sp. dianthi, reaching such a high accumulation level that it was clearly visible after agarose gel electrophoresis of total DNA extracts. FodV1 consists of four double-stranded RNA segments that correspond to a new mycovirus in the Chrysoviridae family. We obtained an isolate of F. oxysporum f. sp. dianthi 116 (116V-) with only a residual level of FodV1 RNA accumulation by single-conidia selection. Compared with 116V-, isolate 116V+ showed significant phenotypic alterations in vegetative growth and virulence. Thus, the presence of a high titer of mycovirus FodV1 was associated with a modified morphology and a reduced growth of the colonies on solid medium, and with a diminished conidiation in liquid medium. Inoculation of four susceptible carnation cultivars with either 116V- or 116V+ showed that the presence of a high titer of FodV1 was also correlated with a significantly reduced virulence of its fungal host. All of the results suggest that FodV1 could be associated with hypovirulence, identifying it as a potential biocontrol agent for Fusarium wilt of carnation. This is the first report of a mycovirus potentially associated with the induction of hypovirulence in the species F. oxysporum.

Control of Fusarium wilt of carnation using organic amendments combined with soil solarization, and report of associated Fusarium species in southern Spain

Fusarium wilt is a disease that restricts carnation (Dianthus caryophyllus L.) yield worldwide. Efficacies in reducing the Fusarium wilt of carnation (FWC), of various types of organic amendments (fresh or pelletized poultry manure, pelletized Brassica carinata and olive residue compost) combined with soil solarization, were compared in two biennial field trials conducted in a greenhouse with a history of carnation monoculture over 8 years. Soil treatments combining organic amendments and soil solarization significantly reduced disease incidence (86–99%) and increased the number of commercial carnation stems by 5–9 times compared to non-treated plots. Twenty-one Fusarium spp. isolates, with different colony morphologies were recovered from soil samples taken in the greenhouse, before the application of treatments in June 2013. Nineteen of them were morphological and molecularly characterized. Additionally, two pathogenicity tests with 17 isolates recovered from greenhouse soils and two isolates recovered from organic amendments were performed. Fusarium species associated with carnation cultivation were identified as Fusarium oxysporum (43%), Fusarium proliferatum (24%), and Fusarium solani (33%). The phylogenetic analysis of the translation elongation factor 1 alpha (EF-1α) region distinguished highly aggressive isolates of F. oxysporum f. sp. dianthi, from low aggressive isolates. The pathogenicity tests showed that FWC has a complex etiology, with several Fusarium spp. identified as causal agents. F. proliferatum and F. solani are associated with carnation wilt for the first time in Spain.

RAPD-PCR technique for molecular detection and diversity of Fusarium oxysporum f.sp. dianthi causing vascular wilt of carnation

Fusarium wilt disease is one of the major economic constraint which hampers carnation cut flower production. Thirteen isolates of Fusarium oxysporum f.sp. dianthi inciting vascular wilt of carnation were evaluated for their molecular characterization using random amplified polymorphic DNA (RAPDPCR). The isolates were categorized based on the morphological characteristics, produced colonies with circular pattern growth. However, differences were obtained in colony colour, shape and size of conidia. The 5.8S-rDNA of ITS region was amplified the specific amplicon size of 398 bp which confirmed the pathogen as Fusarium oxysporum. The molecular polymorphism among isolates were analysed by means of RAPD-PCR and the genetic coefficient matrix derived from the scores of RAPD profile showed that minimum and maximum per cent similarities and diversity among isolates were in the range of 70 to 96 per cent, respectively. The cluster analysis by un-weighted pair-group method with arithmetic average (UPGMA), separated the isolates into four clusters which confirming the genetic diversity among isolates.

Organic amendment, biocontrol agents and soil solarization practice in management of Fusarium wilt of carnation caused by Fusarium oxysporum Schledit. f.sp. dianthi (Prill. and Del.) Snyd. and Hans.

Carnation is severely affected by Fusarium oxysporum f.sp. dianthi which considerably affect the yield and quality of the flowers. Use of organic amendments, biological control agents and soil solarization alone and in combination with antagonists was tried in the present investigation. The results revealed that neem cake and pine needles out of nine organic amendments gave 77.49 and 72.49 per cent disease control with minimum disease incidence. Combined application of two antagonists Trichoderma viride and T. harzianum after 60 days of soil solarization practices registered minimum incidence (16.25%) of the disease followed by T. viride and T. harzianum . The growth characteristics stem length, number of flowers and flower size per plant also increased significantly in these treatments.

Grzyby rodzaju Trichoderma w biologicznej ochronie roślin przed formami specjalnymi Fusarium oxysporum

Effectiveness of Trichoderma species applied into stone-wool and peat infested with Fusarium oxysporum f. sp. radices-lycopersici (Forl) and F. oxysporum f. sp. dianthi (Fod) in the control of Fusarium base rot of tomato and carnation Fusarium wilt was estimated. In three cycles of tomato production in stone-wool Fusarium stem base rot of tomato was observed in all treatments including noninfested combination. Application of Trichoderma spp. into stone-wool resulted in double decrease of total infected plants in the first year of cultivation and 7-fold time in the second year. In the third year of tomato production the disease was not observed. Preplanting application of Trichoderma viride into peat infested with Fod resulted in the significant decrease of Fusarium wilt of carnation. The antagonistic species cause the decrease of Fod population in the substratum. Oceniano przydatność gatunków rodzaju Trichoderma wprowadzonych do wełny mineralnej oraz substratu torfowego, zakażonych przez Fusarium oxysporum f. sp. radicis-lycopersici (Forl) i F. oxysporum f. sp. dianthi (Fod) na rozwój fuzaryjnej zgorzeli pomidorów i fuzariozy naczyniowej goździków. W 3 cyklach uprawy pomidorów w samej wełnie mineralnej, objawy fuzaryjnej zgorzeli wystąpiły we wszystkich kombinacjach, w tym na roślinach kontrolnych bez zakażenia Forl. Dodatek Trichoderma spp. do wełny mineralnej zakażonej przez Forl spowodował około 2-krotne obniżenie liczby porażonych pomidorów w pierwszym roku uprawy i 7-krotne w drugim roku. W trzecim roku nie stwierdzono w nasadzeniu chorych roślin. Zastosowanie T. viride przedwegetacyjnie, do podłoża zakażonego Fod, spowodowało istotne ograniczenie rozwoju fuzariozy naczyniowej. Gatunek antagonistyczny, szczególnie zastosowany w wyższych dawkach, ograniczał rozwój Fod w podłożu.

Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4)

A fluorescent pseudomonad strain P3(4) showing chitinolysis on chitinase detection agar and antagonism against Fusarium oxysporum f.sp dianthi causing vascular wilt of carnation was isolated from pea rhizosphere soil. PCR primers specific for glycosyl hydrolase family 5 (GH5) of Pseudomonas putida isolate KT2440 amplified a 947 bp fragment of the GH5 gene from P3(4). Cloning of this gene into Escherichia coli M15 using an expression vector pQE-30UA and screening on chitin and chitosan detection agar identified one positive clone (Pchi(+) ). Sequence analysis of the cloned insert revealed an open reading frame of 947 nucleotides corresponding to a protein of 315 amino acids with a predicted molecular mass of 38.0 kDa. The deduced amino acid sequence of the open reading frame (gene product/GH) showed 83-84% homology to the GH5 of P. putida strains F1 and KT2440, respectively. The purified enzyme was homogenous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was visualized as single fluorescent band in native gel assay with 4-methylumbelliferyl-N -acetyl-β;-D-glucosaminide and glycol chitosan, respectively. For hydrolysis of 4-nitrophenyl-N -acetyl-β;-D-glucosaminide (pNP-(GlcNAc) and colloidal chitosan, the enzyme had an optimal temperature of 40 °C, and was stable within the temperature range of 10 °C to 40 °C. The enzyme showed an optimal pH of 3.5, with maximum stabilities at 5.0 and 5.5 for hydrolysis of pNP-(GlcNAc) and colloidal chitosan, respectively. Fe(3+) and Cu(2+) stimulated chitinase and chitosanase activities by 74.2 and 51.4%, respectively. The purified GH displayed 70 and 45% inhibition of spore germination of the pathogenic fungi, Fusarium oxysporum f.sp. dianthi and Alternaria solani, respectively.

Effect of Trichoderma asperellum strain T34 on Fusarium wilt and water usage in carnation grown on compost-based growth medium

Suppressive composts are viable alternative growth media for the biological control of plant diseases. To adapt the physical properties of these composts to growth conditions, formulation is sometimes required and consequently their suppressive capacity is reduced. Green house experiments show that plant growth medium based on grape marc compost (compost:peat 1:1, v/v) amended with the biological control agent Trichoderma asperellum strain T34, restores composts’ suppressive capacity against Fusarium wilt of carnation compared with the non-amended medium. Standard chemicals (tocloflos-methyl 50% and captan 85%) used in the cultivation of carnation crops did not improve the suppressive capacity of the growth media or the T34 populations. However, they impaired the stomatal conductance of plants and increased the populations of the pathogen (interaction chemical and T34). Water use measurements of carnation plants, namely stomatal conductance, transpiration and daily water uptake, correlated negatively with Fusarium wilt disease. Plants in T34-amended growth media had higher water demand, regardless of the treatment, which implied their growth could be enhanced by increasing fertirrigation, compared to plants non-amended with T34. In conclusion, the amendment of formulated grape marc compost with T. asperellum T34 improves the suppressive capacity of this growth medium against Fusarium wilt in carnation. It is a better alternative for plant and disease control than the standard chemicals used.

Genetic relatedness of Trichoderma isolates antagonistic against Fusarium oxysporum f.sp. dianthi inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.

In vitro simple determination of resistance to bacterial wilt of carnation

In vitro simple method for determination of resistance to a pathogen of bacterial wilt of carnation (Burkholderia caryophylli) was developed. The bottom of unrooted cuttings of carnation was dipped in bacterial suspension (2.7×102-103cfu/ml) for five min, rinsed with distilled water for ten min, and then incubated with distilled water in test tubes at 30°C. The symptons of bacterial wilt can be visible within two weeks after the inoculation. In the method using unrooted cuttings, the level of resistance of carnation to B. caryophylli was efficiently determined.

Detection of Burkhorderia caryophylli from inoculated carnation by PCR and LAMP (Loop-mediated isothermal amplification)

Burkholderia caryophylli, the pathogen of bacterial wilt of carnation, was detected up to the concentration of 10cfu by PCR and LAMP (Loop-mediated isothermal amplification) using primers based on the domain coding for 16S ribosomal RNA. LAMP was a simple and easy method to detect the bacteria compared with PCR, because LAMP reaction was performed in a shorter time than PCR. The bacteria can be detected by LAMP using materials derived from stuck wooden tooth-pick into the stem of cuttings inoculated with B. caryophilli.

Variability of pollen grain features: a plant strategy to maximize reproductive fitness in two species of Dianthus ?

Pollen polymorphism was observed in populations of Dianthus caryophyllus and Dianthus chinensis within species and within genotypes. Germination capacity and vigor of pollen tube growth are correlated with grain size, as demonstrated using pollen culture in an extract of cultured Fusarium oxysporum Schlecht. f. sp. dianthi – the causative agent of carnation wilt. Larger grains exhibited higher germination frequency, suggesting adaptation for adverse conditions. Pollen tube growth rate, however, was negatively correlated with pollen size. Since pollen tube growth rate is typically associated with the vigor of the resulting sporophyte, smaller pollen grains may have a selective advantage and produce more vigorous progeny. The variability of germination response among various size classes of pollen grains suggests the occurrence of multiple pollen size classes as a survival strategy. The study also indicates a relationship between such pollen grain features as grain size and pollen tube growth rate as a strategy to maximize reproductive fitness.

Influence of Pythium oligandrum on population of Fusarium oxysporum f.sp. dianthi and development of Fusarium wilt of carnation

Relationship between initial oospore number of Pythium oligandrum, mode of the mycoparasite application and population<br />dynamics of Fusarium oxysporum f.sp. dianthi and development of Fusarium wilt of carnation was studied. Mixing of<br />oospores with peat 10 days before carnation planting resulted in strong inhibition of the pathogen development. Number<br />of colony forming units of the pathogen decreased at least 3 times. Application of P. oligandrum resulted in suppression<br />of Fusarium wilt development, especially when the mycoparasite was applied at dose 100 oospore/g of peat.

Detection of Fusarium oxysporum f. sp. dianthi in Carnation Tissue by PCR Amplification of Transposon Insertions

ABSTRACT Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transposable elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi strains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolates of race 4. Genomic DNAs flanking race-associated insertion sites of Fot1 (from races 1, 2, and 8) or impala (from race 4) were amplified by the inverse polymerase chain reaction (PCR) technique. These regions were cloned and sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DNA as template in PCR experiments, primer pairs generated amplification products of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with genomic DNA belonging to races 1 and 8, 2, or 4, single amplimers were generated, allowing clear race determination of the isolate tested. PCR was successfully performed on DNA extracted from susceptible carnation cv. Indios infected with isolates representative of races 1, 2, 4, and 8.

Stability of transformed antagonistic Fusarium oxysporum strains in vitro and in soil microcosms

AbstractThe stability of a genetically modified strain of Fusarium oxysporum used as antagonist against phytopathogenic formae speciales of F. oxysporum was evaluated both in vitro and in microcosm assays. The Escherichia coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance, was introduced by genetic transformation into a recipient strain marked by benomyl resistance and a dark red pigmentation. Hybridization with the complete plasmid suggested that the integration had generally occurred in a multiple‐tandem array at multiple sites. Among nine independent transformants tested, only three of them were mitotically stable after four rounds of vegetative growth with no selective pressure, while six showed various changes in the integration pattern. One transformant had lost the ability to grow in the presence of hygromycin B. In soil microcosms all the transformants maintained the hygromycin B resistant phenotype, but six of them showed rearrangement of transforming DNA. Only one strain (coded T26.40) underwent no obvious rearrangement both after in vitro growth and after recovery from the soil microcosm. The nine transformants were used in three biological control experiments against Fusarium wilt of carnation in comparison to two untransformed reference strains and to the recipient mutant. A high degree of variability in the biocontrol activity was observed throughout the experiments and only transformant T26.40 consistently controlled the incidence of disease. The results are discussed in relation to risk assessment of the release of transgenic antagonistic fungi.