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

Background Carnation, 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. Results The 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. Conclusions The investigation suggested that Trichoderma spp. could be a potential biocontrol agent to mitigate Fusarium wilt in carnation and improve production quality


Background
Fusarium wilt caused by Fusarium oxysporum f. sp.dianthi (Fod) is a soil-borne fungal disease that affects a wide range of economically important plants worldwide, from crops to ornamental flowers (Cer et al. 2022).The first report of Fod races in Iran was in 2011 (Zahiri et al. 2013).Carnation is among the most popular commercially cut flower, after roses and chrysanthemums, due to its varied colors and exquisite shape (Migheli et al. 1998).The most devastating pathogenic fungi affecting carnation (Dianthus caryophyllus L.) and a major limiting factor for cultivation and export in Iran and worldwide is Fusarium wilt, caused by Fod (Zandyavari et al. 2013).There are various methods for controlling this disease, but using Trichoderma strains for biocontrol is considered safer and more preferable (Coşkuntuna and Özer 2008).
The nonpathogenic and saprophytic filamentous fungus Trichoderma is a joint antagonist agent capable of producing a variety of hydrolytic enzymes and antifungal metabolites that can colonize and suppress many phytopathogens, such as those from Rhizoctonia, Pythium, Phytophthora, Sclerotium, and Fusarium genera (Filizola et al. 2019).Trichoderma spp.have been characterized as promising BCAs against plant nematodes and soil-borne pathogens, such as F. oxysporum, Rhizoctonia solani, and Botrytis cinerea (Mukhopadhyay and Kumar 2020), and they are considered the potential biocontrol alternatives for plant disease management due to the diverse range of a plethora of metabolites they possess (Phoka et al. 2020).They contribute to inhibiting up to 80% of economically important plant pathogens (Sánchez-Montesinos et al. 2020).Several investigations have found that Trichoderma spp.exhibit a variety of biocontrol mechanisms.These mechanisms include mycoparasitism, antibiosis, competition for nutrients and space, stress tolerance via increased root and plant development, inorganic nutrient solubilization and sequestration, systemic acquired resistance, and enzyme inactivation (Guzmán-Guzmán et al. 2023).
Biocontrol is an environmentally friendly approach to managing plant diseases without leaving chemical residues on living organisms (Alvarado-Marchena and Rivera-Méndez 2016).With the global challenge of food scarcity, efficient BCAs are in demand for improving crop production and disease management.Trichoderma is a multipurpose BCA with multiple antagonistic properties that can mitigate many plant diseases and promote plant growth (Woo et al. 2023).This study was carried out to evaluate the antagonistic potential of Trichoderma harzianum and T. viride against Fusarium wilt disease in carnation flowers.

Pathogen isolation
Soil samples were collected from the Tehran, Markazi, and Fars provinces of Iran for isolation.Ninety-six isolates, used in this study, were maintained in the culture collections of the Department of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University (Table 1).These isolates were identified as Fod based on a pathogenicity test (Zandyavari et al. 2013).The Fod isolates were cultured on PDA media for 5 days and then purified using the single-spore method.

Isolation of antagonists
Twenty-five rhizospheres soil samples containing antagonistic fungi, including Trichoderma, were collected from carnation fields in greenhouses.The soil samples weighing 1 g were obtained using the serial dilution technique (Marraschi et al. 2019).Trichoderma selective medium (TSM) was used to isolate the Trichoderma from the samples (Marraschi et al. 2019).The colonies emerged in the Petri plates and were subsequently purified using the hyphae tip isolation technique.For this, 1 ml of the 10-3 dilution was plated on the aforementioned selective medium.

Biocontrol tests Evaluation of Trichoderma spp. antifungal effect against Fod in vitro
This experiment was conducted on PDA medium using the dual culture method as described by Unartngam et al. 2020.Ten isolates of T. harzianum and T. viride were cultivated with the pathogen isolates on PDA for a week.Disks measuring 5 mm in diameter from the growing borders of the colonies were selected and transferred to other PDA dishes.Two disks of Trichoderma spp.and the plant pathogen mycelium were placed opposite each other near the periphery of the Petri plate and incubated at room temperature for 7 days.In control plate, only the pathogen was placed as no antifungal isolate was applied.The experiment was replicated three times, and the proportion of mycelium inhibition was measured using the method described by Dugassa et al. 2021.where I = percentage inhibition; R 1 = radial growth of pathogen in control plate (cm); and R 2 = radial growth of pathogen in dual culture plate (cm).

Evaluation of the volatile compounds
Properties of volatile substances secreted by Trichoderma spp. on the mycelial growth of Fod using the technique of paired Petri dishes described by Mahalakshmi and Raja 2013 were evaluated.Disks measuring 9 mm in diameter, obtained from 4 days cultured antagonistic isolates, were placed on PDA plates.The bottom of each PDA dish, inoculated with the mycelia disk of Fod, was then placed on top of Petri dishes flipped over Trichoderma spp.dishes, and the setup was incubated at 25 °C for 4 days.The PDA plates were also inoculated with Fod alone and paired without Trichoderma spp. as a control.Three replications were conducted for each isolate.After the incubation period, the mycelial growth of the pathogen was measured and expressed as a percentage of inhibition over the control.The ability of volatile compounds to control pathogen growth was evaluated using the mentioned formula (Dugassa et al.

Competitive saprophytic ability (CSA) assay
To conduct the colonization success assay (CSA) experiment with Trichoderma spp.isolates, the procedure described by Mahalakshmi and Raja 2013 was applied.All isolates of Trichoderma spp.were cultured on molasses yeast medium, and the conidial concentration was adjusted to 106 per gram, which was then mixed with unsterilized soil and placed in plastic pots.Each pot was prepared with 21-cm segments of clean paddy straw buried in the soil.The plastic pots were covered and incubated in a dark environment for a week to maintain moisture.
After the incubation period, twenty pieces of each isolate were retrieved from the plastic pots, washed, and disinfected using NaCl 2 and ethanol.The desired pieces were then plated on the TSM and incubated at 25 °C for 5 days.The experiment was replicated three times for each isolate, as mentioned in Marraschi et al. 2019.The results of the CSA were calculated using the same formula:

Biocontrol evaluation under greenhouse
The effectiveness of T. harzianum and T. viride isolates in controlling Fusarium wilt in carnation cultivated in pots was evaluated under greenhouse conditions, following the methods outlined in Locke et al. 1985 with some modifications.For this experiment, standard carnation rendezvous cuttings were selected due to their high susceptibility to Fusarium wilt.The cuttings were individually planted in pots containing a sterilized soil mixture of sand, peat, and silt (2:1:2 by volume) and kept in a greenhouse at 25 °C with natural light and 55-60% R.H.The treatments were 106 CFU/ml spore suspension of each antagonist isolate to each pot, along with 106 CFU/ml spore suspension of Fod isolates.A total of 288 pots were used to evaluate the antagonist isolates, and twelve pots were used as control (without antagonist isolates against Fod).After an incubation period of 90 days, the occurrence of Fusarium wilt in the carnation was assessed and recorded.

Statistical analysis
The data were statistically analyzed using analysis of variance (ANOVA), followed by post hoc analysis.Duncan's multiple range test was used to determine the significance of the treatments compared to the control, and a significance level of p < 0.05 was applied.The greenhouse treatment was analyzed using the randomized complete block design (RCBD) test.The data were subjected to normality and heterogeneity tests before analysis of variance.All statistical analyses were done using SPSS software (version 26) (Levesque 2007).

Molecular identification and phylogenetic analysis
The genomic DNA of the selected isolates was amplified using ITS1 and ITS4 primers targeting the rDNA region.
The ITS PCR was utilized to identify polymorphisms within the ITS region of rDNA among the Fusarium isolates.The results demonstrated that 96 Iranian Fusarium isolates showed a band of approximately 600 bp in size (with a fragment size ranging from 550 to 578 bp) on a 1.2% agarose gel, and they were characterized as Fod (Fig. 1).The twenty Trichoderma spp.isolates displayed amplified products at approximately 900 bp (Fig. 2).
All amplified products generated with the RAPD primers showed polymorphic and distinguishable bands, indicating the genetic diversity of Trichoderma isolates.Among the twenty Trichoderma isolates, seven primers produced 104 reproducible polymorphic bands, ranging in size from approximately 100 to 3200 bp (Table 3).The RAPD profiles showed that primers A11 and AA04 generated the highest number of bands, ranging from almost 410 to 2000 bp and 400 to 3000 bp, respectively.Moreover, the molecular characterization of T. viride and T. harzianum isolated from Iranian soils revealed polymorphic bands ranging from 296 to 3300 bp, with primer A05 producing the highest number of bands among the RAPD primers.
Cluster analysis of the data was performed using the similarity matrix to identify relationships among the isolates (Additional file 1: Table S4).The similarity   coefficients ranged from 0.15 to 1.00 among the isolates.The similarity matrix revealed that isolates Th1 and Tv6 were genetically distinct, showing only 15% similarity.The dendrogram was generated using the UPGMA and NTSYSpc software based on the similarity matrix (Fig. 3).According to the results obtained, all twenty Trichoderma spp.isolates could be grouped into two major clusters: One cluster comprised of T. viride and the other of T. harzianum.Different numbers of isolates were present in each sub-cluster.

Biocontrol activity of Trichoderma spp. in vitro
This study examined twenty isolates of T. harzianum and T. viride (ten isolates of each species) for their antifungal activity against the Iranian soil-borne pathogen, Fod.These antagonists demonstrated varying degrees of antagonistic activity isolates against Fusarium wilt in dual culture plates, volatile compounds, and CSA (competitive saprophytic ability) tests.
Among the tested isolates in this study, T. harzianum (Th1) revealed the highest mycelia growth inhibition proportion of 58%, followed by Th7 with 56.5% and Th2 with 48.6% inhibition of mycelia growth.The lowest percentage of mycelia growth inhibition was observed in T. viride (Tv3) and Th5, with only 15% (Table 4) showing a significant variation (P ≤ 0.05).Figure 4 demonstrates the control of Fusarium mycelium growth by Trichoderma spp.
The results revealed a direct correlation between the colonization proportion of Trichoderma spp.and their CSA ability.Isolates Th1 and Th7 demonstrated the highest level of CSA.
The fungi recovered from plant material buried in soil fields showed the ability to colonize these substrates as competitive saprophytes.Similarly, the greenhouse evaluation study revealed significant differences between T. harzianum, T. viride, and control treatments.These differences were observed in disease symptoms indications, with either slight disease symptoms or complete eradication of the pathogen in the carnation stems or vascular system.After 90 days of inoculation, the Fod control treatment resulted in complete mortality (Fig. 6).

Discussion
Fusarium wilt of carnation caused by Fod is one of the challenging soil-borne pathogens that devastate carnation cultivation around the world (Wolcan et al. 2016).Mycoparasitism, nutrient competition, induced plant    (1997), who studied and identified the genetic diversity in F. oxysporum f. sp.dianthi and Fusarium redolens f. sp.dianthi isolates.Distinguishing features in the morphology of Trichoderma strains, such as copious conidial pigments in white or bright green, along with conidiophores that are repetitively branched and lack clear definition, enable their recognition at the genus level (Rifai 1969).The BCAs Trichoderma spp.have been widely considered excellent alternative control agents against plant pathogens such as Fusarium strains (Filizola et al. 2019), brown spot of rice (Charoenrak and Chamswarng 2015), Phytophthora infestans, the agent of potato late blight (García-Núñez et al. 2017), and other plant pathogens like Fusarium wilt on carnation (Marraschi et al. 2019).Trichoderma spp.can adapt to various living environmental factors and conditions (García-Núñez et al. 2017).The present study revealed that Fusarium mycelium was effectively controlled by T. harzianum (Th1) exhibited the highest proportion of mycelial growth inhibition at 58%, followed by Th7 with 56.5%, and Th2 with 48.6% inhibition.Pratibha (2000) reported that T. harzianum had inhibited the mycelia growth of Fod.Similarly, it has been reported in India that T. harzianum, T. viride,   and T. ressei showed significant efficiency against Fod (Mahalakshmi and Raja 2013).The antagonistic impact of Trichoderma spp.against other plant pathogens has also been reported (Gupta and Misra 2009).
There was a varied efficiency of volatile compounds in inhabiting the growth of Fod, particularly the volatile compounds from T. harzianum isolates Th1, Th7, and Th2 demonstrated the most significant growth inhibition against Fod.The previous studies have shown that Trichoderma spp.produce both volatile and non-volatile compounds that can affect the growth of different plant pathogenic fungi (Mahalakshmi and Raja 2013).In line with that, Mahalakshmi and Raja (2013) reported that T. harzianum and T. viride isolates exhibited biological efficacy against the vascular wilt pathogen in carnation.Khaledi and Taheri (2016) found that volatile compounds production is one of the major antagonistic mechanisms against Macrophomina phaseolina pathogenic fungi.Similarly, volatile and non-volatile compounds from T. harzianum and T. viride have been reported to be associated in controlling Fusarium spp.and Alternaria spp.(Meena et al. 2017).
A clear correlation between the colonization proportion of Trichoderma spp.and their ability was to compete for space and nutrients.This correlation between colonization and CSA was confirmed and reported by García-Núñez et al. (2017).It was clear that these fungi have the capacity to thrive and compete in the soil environment from the recovered plant material from soil.Locke et al. (1985) reported that the increased antagonistic potential of T. viride isolates was attributed to enhance CSA.Furthermore, Thiruvudainambi et al. (2010) demonstrated that the percentage of mycelia growth among Sclerotium rolfsii isolates declined due to the robust CSA exhibited by T. viride isolates.Moreover, several investigations have reported a correlation between the proportion of colonization among Trichoderma spp.isolates against Fod and other pathogens with their CSA activity (Dugassa et al. 2021).Hermosa et al. (2000) reported that conducting experiments in natural environments is essential to validate further the antagonistic effect of a biocontrol strain selected through a laboratory protocol for potential biocontrol against plant pathogens.
A significant impact of Trichoderma treatments on disease symptoms and pathogen eradication was observed in the greenhouse experiment; these findings imply that Trichoderma spp.isolates in this study profoundly eliminate the Fusarium wilt in carnation under laboratory conditions and greenhouse setting.Comparable results were obtained in the greenhouse, where Trichoderma spp.effectively controlled the Fusarium wilt of chrysanthemums (Locke et al. 1985).According to a report by Fattahi (2014), it was confirmed that the cultivation of greenhouse-grown carnation in Iran's Markazi province was significantly impacted by Fusarium wilt.The versatile qualities of Trichoderma spp., from effective defense mechanisms and the highest canalizations and competition in their habitats through potent degradation enzymes, make them promising biocontrol fungicides and potential candidates for industrial production (Schuster and Schmoll. 2010).The study by Martinez (2023) highlighted the use of carrier substances, drying techniques, and microencapsulation to create stable, high-performing products from Trichoderma spp.for developing effective and life shelf fungicides.Trichoderma spp.are complex plant hosts that communicate with their hosts and provide multifaceted benefits.They function as a plant growth promoter, inducer of local and systemic defense responses against biotic and abiotic stresses and activator of transcriptional memory affecting future stress responses (Woo et al. 2023).

Conclusion
The potential of T. harzianum and T. viride isolates as BCAs against Fusarium wilt caused by Fod in carnation was evaluated.In vitro assays showed isolates Th1, Th7, and Th2 of T. harzianum and Tv5, Tv4, and Tv7 of T. viride exhibited the highest antifungal activity against the pathogen.Treatments with these efficient isolates in the greenhouse led to significant Fusarium wilt reduction or eradication in carnation.The study found that Trichoderma isolates are a highly effective biological control agent for managing this disease.Further field studies to establish their efficacy before implementing their use in agriculture are recommended.

Fig. 3
Fig.3The genetic relationship between the twenty Trichoderma isolates based on RAPD analysis
resistance, and the synthesis of antibiotic and hydrolytic enzymes biocontrol mechanisms of Trichoderma spp.make them an excellent candidate for biocontrol application against many plant pathogens(Manzar et al. 2022).Our investigation aimed to explore the potential of Trichoderma isolates from Iranian soils for combating the Fusarium wilt of carnation.The results confirm the characterization of these isolates as Fod.These results align with Baayen et al.

Fig. 5
Fig. 5 The inhibitory effect of volatile compounds on the pathogen growth

Fig. 6
Fig. 6 Comparison of vascular structures in an unprotected plant treated with an antagonist (A) and a protected plant (B)

Table 1
Trichoderma spp.and Fod isolates from three

Table 2
RAPD primers and their sequences

Table 3
Analysis of the polymorphism obtained with seven RAPD markers in 20 Trichoderma isolates

Table 4
Antifungal activity of Trichoderma spp.against the mycelia growth of the pathogen *The mean of three

Table 5
Inhibitory effect of the volatile component of Trichoderma spp.against Fod growth in vitro

Table 6
The competitive saprophytic ability (CSA) of T. harzianum and T. viride isolates *The mean of three replications