Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 in banana plantations in Türkiye

Summary. Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 ( Foc TR4), is an important disease for banana production. Presence and prevalence of Foc TR4 in banana plantations on the Mediterranean coast of Türkiye were assessed during 2018 to 2020 in a total of 117 banana plantations in open fields and protected plastic greenhouses. Rhizome, pseudostem, and root samples were taken from plants showing typical symptoms associated to the disease and from suspected affected plants. Fungi were isolated from the plant internal tissues, and Fusarium oxysporum -like colonies were sub-cultured for further analyses. Phylogenetic analyses of 36 isolates showed that they belonged to four different Fusarium species: F. musae, F. oxysporum, F. sac-chari , and F. solani . Eight representative F. oxysporum isolates were identified as Foc-TR4 by specific PCR and qPCR tests. Pathogenicity tests were carried out on tissue-cultured ‘Cavendish’ type banana seedlings (‘Grand Naine’) for 36 Fusarium isolates, and their virulence was assessed based on the internal necrosis observed in the rhizomes. Approx. 40 to 65 d after inoculations, Foc TR4, F. oxysporum , and F. sacchari isolates caused severe to mild necroses in the seedling rhizomes. This is the first report of F. sacchari associated with root and collar rot of bananas in Türkiye. This study showed that Fusarium wilt caused by Foc TR4 is present, but at low incidence (6.8%) in Turkish banana plantations.


INTRODUCTION
Bananas and plantains (Musa spp.) are important agricultural products in tropical and subtropical regions.Banana plantations are located on the coastline of the Mediterranean Region of Türkiye, and 883,455 tons of sweet bananas are produced annually from approx.11,000 ha (Anonymous, 2022).This production does not meet the domestic consumption, and 20% of domestic consumption is imported from other banana-producing countries, including Costa Rica and Ecuador (Anonymous, 2021).Fusarium wilt, caused by Fusarium oxysporum Schlecht.f. sp.cubense (E.F.Smith) Snyder and Hansen (Foc), is the most important fungal disease in bananaproducing countries, due to the lack of practical management methods and the severe economic losses this disease can cause.Foc first infects banana plants via root hairs, then develops in the internal corm tissues, progressing to the xylem vessels and activating xylemsecreted genes that trigger initial symptoms (Dong et al., 2016;Czislowski et al., 2021).Water transport in the xylem is disrupted due to the conidial and mycelial mats produced by the pathogen, and by host substances such as gums, and mechanical barriers produced to prevent systemic spread of the fungus.Enzymes and toxins produced by Foc cause the host leaves to turn yellow and the lower leaves to droop (Stover, 1962).
Fusarium wilt of banana was first reported in Brisbane, Australia, in 1874 but reached severe levels in Panama and Costa Rica in the 1890s (Pegg et al., 2019).Destruction of the Gros Michel cultivar (susceptible to race 1 of Foc) by the fungus in Latin America and the Caribbean has made it one of the most important fungal pathogens wherever bananas are grown.Since the early 1960s, Fusarium wilt was not of concern due to adoption of the Foc resistant banana cultivar 'Cavendish'.However, despite the use of resistant 'Cavendish'-related cultivars, the disease re-appeared in the 1990s in Taiwan, Indonesia and Malaysia, caused by the new physiological race, Tropical Race 4 (FocTR4) (Ploetz, 2006).To date, four physiological races of Foc have been described to cause Fusarium wilt, including Race 1 in 'Gros Michel' and 'Silk'; Race 2 in 'Bluggoe'; Race 3 in Heliconia spp., and Race 4 in all commercial banana cultivars.In addition, the subtropical race of the pathogen (FocSTR4) was found to be ineffective in the tropics or in areas where optimum banana growth conditions occur.
As with other soil-borne plant pathogenic fungi, managing FocTR4 is challenging, and practical control has not yet been found for eradicating the pathogen from soil.Foc has long persistence in the soil (more than 20 years), due to its ability to survive as a saprophyte in plant residues and to produce chlamydospores.As these inocula are pushed deeper into the soil with tillage, eliminating the pathogen from infested areas becomes almost impossible (Stover, 1962).Solarization is ineffective in eradicating this pathogen when applied alone, because of its inability to penetrate deep into the soil (Herbert and Marx, 1990).In addition, using this method in large areas is difficult and costly.Breeding for resistance is ongoing, but a banana cultivar with high commercial potential and complete resistance has not yet been produced, as was previously the case for the Foc1 and Foc2 resistant 'Cavendish' (Dita et al., 2018).
Fusarium oxysporum is a large species complex including saprophytes, endophytes, plant and human pathogens; it also has 143 formae speciales (106 of them are well-documented) and 25 physiological races (including FocTR4) from monocotyledon and dicotyledon host plants (Edel-Hermann and Lecomte, 2019).Formerly, Fusarium species have been identified based on their morphological characteristics, which primarily include asexual reproductive structures (chlamydospores, conidiophores, macro-and microconidia, and phialides) and colony morphology (Fourie et al., 2009).However, molecular tools and phylogenetic analyses supported by DNA gene sequencing, as well as pathogenicity tests, have shown that these features are insufficient for species identification.Since it is not possible to discriminate pathogenic isolates (or physiological races) using morphology and microscopy, precise diagnostic procedures (e.g.PCR, gene sequencing, VCG tests) and pathogenicity tests must be used for accurate identification.In the last 15 years, commercial diagnostic kits and PCR primers that amplify specific regions in genomic DNA of these races have been designed for molecular identifications.Lin et al. (2009) followed the RAPD marker technique to design specific primers for identifying tropical race 4 using 96 FocTR4 isolates from Taiwan.Their primer (Foc1/Foc2) amplifies a specific 242bp gene product in Foc genomic DNA.Dita et al. (2010) designed FocTR4-F/FocTR4-R primers to identify FocTR4 strains using single nucleotide polymorphism in the IGS region.These primers have been widely used for monitoring the disease and in pathogen identification studies.Some commercial diagnostic kits containing pathogenpositive DNA have also been developed for qPCR ampli-fication, and have been used in studies aimed at disease monitoring in banana-producing countries (Dale et al., 2017;García-Bastidas et al., 2019).
If the pathogen has not appeared in a country, the most effective control measure is to exclude the pathogen by implementing strict quarantine measures.When the pathogen is detected in a limited area, it is necessary to contain and eradicate it before becoming widespread.Pathogen tracing and disease monitoring with regular surveys, and accurate identification procedures, are essential.Fusarium wilt of banana was first reported in Türkiye was in 2018.FocTR4 was detected in three protected plastic covered greenhouses after identification using specific PCR primers and pathogenicity tests (Özarslandan and Akgül, 2020).However, a more comprehensive study is required to determine prevalence of Fusarium wilt in Türkiye.
The aim of the present study was to determine the prevalence of Fusarium wilt caused by FocTR4 in openfield and plastic-covered-greenhouse banana plantations in Türkiye.Knowledge of FocTR4 prevalence in these areas would provide a basis for appropriate quarantine measurements, and for further investigations on disease management and screening of local banana cultivars for resistance breeding.

Survey, sample collection and isolation of fungi
A survey was carried out in protected plastic, greenhouses (n = 72) and open field banana plantations (n = 45) located in Seyhan, Yüreğir (Adana province), Alanya, Gazipaşa (Antalya province), Arsuz (Hatay), and Anamur, Bozyazı, Erdemli, Silifke, Tarsus districts (Mersin province), in the Mediterranean Region of Türkiye (Figure 1).A total of 117 plantations (approx.total area 76 ha) were inspected, from March 2018 to December 2020.Individual banana the plants were examined for general appearance, and rhizome, pseudostem, and root samples were collected from those with typical disease symptoms (Figure 2).These samples were placed in paper bags and then in an ice box, and transported to a laboratory for further processing.
Small sections (5 to 8 cm each) of symptomatic pseudostem, corm, and root tissues were dissected from each sample, and then surface sterilized with 2.5% sodium hypochlorite solution (>5% active chlorine) for 3 min, then rinsed twice in sterile distilled water.The internal tissues (4 to 6 mm) from each section were aseptically placed onto ¼ strength Potato Dextrose Agar (PDA; CondaLab) amended with streptomycin sulfate (250 µg mL -1 ), and the culture were incubated at 25°C for 4-5 d.Twenty Petri dishes were used isolations from tissues from each banana plantation.After morphological and microscopic examinations, Fusarium oxysporum-like colonies were transferred by single-conidium isolation techniques onto fresh PDA plates, backed up on sterile green banana leaf cultures, and were stored at -20°C (Seifert, 1996).Among the F. oxysporum-like fungi, a representative sample of 36 isolates was selected for further pathogenicity testing and gene sequencing.These isolates were tentatively identified using cultural and microscopical characteristics (fluffy mycelia, simple short phialides, and pale violet or pinkish colony colour), as described by Nelson (1983) and Leslie and Summerell (2006).FocTR4 prevalence (%) was calculat- ed based on the number of greenhouse or banana fields (where FocTR4 detected and the total number of banana plantations (surveyed).The isolation frequency (%) of all fungi in each plantation was estimated using 140 tissue pieces plated (20 Petri plates, seven tissue fragments each) from symptomatic plants.

Molecular identification of Fusarium isolates
Thirty-six F. oxysporum-like isolates (tentatively identified based on their simple short phialides on hyphae, salmon or pale violet colony colour after 3 weeks incubation on PDA at 24°C in the dark) were selected for further molecular identification.Isolates were grown on PDA at 24°C for 8-10 d in the dark.Fresh aerial mycelia were collected by scraping the colonies with a sterile scalpel, and the mycelia were placed in sterile Eppendorf tubes containing 2% CTAB buffer.The following extraction steps were then followed, as described by O'Donnell et al. (1998).For gene sequencing, translation elongation factor 1α (TEF-1α) and intergenic spacer (IGS) region (nuclear ribosomal operon) genes were amplified using PCR using, respectively, the EF1/EF2 primer (O'Donnell et al., 1998) and FocTR4F/FocTR4R primer (Dita et al., 2010).Each PCR reaction mixture contained 5 μL of buffer (10× Green Buffer, DreamTaq Green DNA Polymerase, Thermo Scientific™), 2 μL of the dNTPs mixture (10 mM each, Thermo Scientific™), 0.5 μL of forward and reverse primers (stock concentration: 10 pmol•μL -1 ), 0.25 μL of Taq polymerase (DreamTaq Green DNA Polymerase, Thermo Scientific™), 39.75 μL of PCR grade water and 1 μL of genomic DNA (approx.100 ng•μL -1 ).PCR amplifications were carried out in a thermocycler (Simpli-Amp A24811™ Thermal Cycler, Applied Biosystems), using the conditions specified in Table 1.PCR products were separated by gel electrophoresis in 1.5% agarose (Sigma) gels in 1× Tris-Acetic acid-EDTA (TAE) buffer, to check DNA band size and quality.These products were then sequenced by Macrogen Co. (South Korea), and the sequences were compared with those deposited in the NCBI GenBank database using the BLAST tool (version 2.0; National Center for Biotechnology Information, US National Institutes of Health).TEF-1α and IGS sequences obtained were deposited to the NCBI GenBank.
FocTR4 identification was also confirmed by Real-Time PCR using SYBR® Green technology (Clear Detections TR4 Kit™).For each isolate, 5 µL of genomic DNA was added to 200 µL capacity thermocycler tubes each containing 15 µL of Clear Detections qPCR master mix, then vortexed for 20 sec. of PCR-grade water (5 µL) and FocTR4 genomic DNA (provided by the manufacturer) were then added to other PCR tubes to confirm negative and positive amplifications as controls.The thermal cycler (Roche Light Cycler 480™) was set according to the conditions outlined in Table 1, and whether the isolates were FocTR4 was determined according to the obtained cycle thresholds (Ct).To avoid missing weak positive amplifications due to DNA quality and other factors, melting-curve analysis was also carried out, and amplifications around 75°C were considered primer dimers.The molecular identification tests were each repeated once.
The phylogenetic analysis was carried out using data from the translation elongation factor-1α gene.The data set was constructed using reference sequences (Table 2) from relevant publications (Maryani et al., 2019a;Crous et al., 2021;Tava et al., 2021).The sequences were aligned using the algorithm in Muscle, and a maximum likelihood tree was constructed using MEGA-X software with the Hasegawa-Kishino-Yano model (Hasegawa et al., 1985).Geejayessia zealandica (= F. zealandicum) CBS isolate 111.93 was used as a root taxon, and node support was estimated by bootstrap analysis on 1000 random trees.

Pathogenicity tests
The 36 selected Fusarium isolates were used in pathogenicity tests on banana seedlings, as described by Thangavelu et al. (2019).The isolates were grown on PDA for 7-8 d at 25°C in the dark.The mature cultures were then flooded with sterile distilled water, and mycelia were scraped with a sterile plastic needle to dislodge macro and microconidia.The conidium suspensions were each filtered through two layers of sterilized cheesecloth, and the concentration of resulting conidium suspension was adjusted to 10 6 conidia mL -1 , after enumeration with a Thoma® slide under a light microscope.Banana seedlings ('Grand Naine' at 4 to 6 leaf stage, produced by tissue culture), were uprooted from trays; their roots were slightly trimmed and then dipped into the conidium suspensions of respective isolates for 10 min.The seedlings were then planted in plastic pots (15 cm diam.) containing sterile peat moss, sand and perlite mixture (1:1:1 v/v/v), and were placed in climate controlled greenhouses (at 27°C, 85% relative humidity, 12 h illumination).The inoculated plants were grown for 65 d, and the pathogenicity of the isolates was assessed using a 0 to 3 severity scale, based on the discolouration of the each plant rhizome and whole plant wilting, as described by Li et al. (2015).Disease severity was calculated using the formula of Towsend and Heuberger ( 1943

Statistical analyses
Analysis of variance (ANOVA) were carried out on disease severity data (mean lesion lengths in two experiments), and the data were checked for normality.Means were compared using Fisher's least significant difference (LSD) test at P ≤ 0.05 (Gomez and Gomez, 1984).

Fungal isolations, identification of FocTR4, and disease severity assessments
In the surveyed banana plantations, 12 fungal genera, including Alternaria, Aspergillus, Cladosporium, Epicoccum, Fusarium, Macrophomina, Nigrospora, Penicillium, Pythium, Phytophthora, Rhizoctonia, and Trichoderma (based on ITS sequencing and simple BLAST searches), were obtained from symptomatic and asymptomatic banana plants.Among these fungi, Fusarium was the most commonly isolated genus, obtained from 96% (112 of 117 plantations) of the total plantations assessed.According to cultural morphological characteristics of isolates (fluffy mycelia, simple short phialides, and pale violet or pinkish colonies) and microscopic features (short or long simple phialides, macro-and microconidium shapes, chlamydospore production), 36 Fusarium isolates were selected for further identification studies.
The BLAST results were confirmed by the clustering of the isolates with respective reference sequences of F. musae (F31), F. oxysporum (CAV794, NRRL_26029), F. sacchari (NRRL_13999), and F. solani (CBS 102429) in the phylogenetic tree.However, these methods could not differentiate FocTR4 from the closely related Fusarium oxysporum isolates.
The conventional PCR tests showed that eight of the F. oxysporum isolates belonged to the tropical race four, so 463 bp DNA bands were observed with agarose gel electrophoresis (Figure 4).However, genomic DNAs from the remaining 28 isolates could not be amplified using these specific primers.
The real-time PCR tests using a FocTR4-specific diagnostic kit agreed with the conventional PCR results.On average, while the CT value average for the reference DNA provided by the Clear Detection™ commercial kit was 21.59, these values varied between 24.97 and 31.63 in eight isolates suspected to be FocTR4 (Figure 5).No amplification was recorded for DNA of the other Fusarium isolates (including the water control), and their Ct values were greater than 36 (Table 3).

Pathogenicity tests
Approx.40 d after inoculating 'Grand Naine' banana seedlings with the Fusarium isolates, some plants showed yellowing of the lower leaves (Figure 6, a  and b).Plants inoculated with two Fusarium solani isolates (BMAE41 and BMAE43), F. musae (BMAE3MM), or the non-inoculated control plants, did not develop disease symptoms (Figure 6 j).Approx.50 d post inoculation, yellowing symptoms progressed to the upper leaves, while the lower leaves wilted and dried thoroughly.Two weeks after these symptoms appeared, plants inoculated with isolates identified as FocTR4 wilted and died, while plants inoculated with the other isolates continued to live for approx.20 d.Rhizome necroses started from the pith tissues in FocTR4 inoculated plants (Figure 6 c to f), this progressed from the cortex to the centres in F. oxysporum and F. sacchari inoculated plants (Figure 6 g and h).According to the evaluation scale of Li et al., (2015), F. sacchari and F. oxysporum caused rhizome necrosis and seedling death at rates from 33.3% to 83.3%, while no symptoms were observed from F. solani (BMAE41MM and BMAE43MM) or F. musae (BMAE-3MM) isolates and the sterile water inoculated controls (Figure 6 j).Disease severity was greater (66.7-91.7%) in plants inoculated with FocTR4 isolates (Figure 7).Fusarium isolates were re-isolated from internal rhizome tis-sues of these plants.Fusarium solani isolates were re-isolated only from the hairy roots (not rhizomes), while no Fusarium colonies were obtained from the rhizomes of the non-inoculated control plants.Based on the overall averages of all isolates from each species, F. oxysporum f. sp.cubense TR4 caused the most severe rhizome necrosis (74.0%), followed by F. sacchari (61.5%) and F. oxysporum (58.3%).

DISCUSSION
Fusarium wilt of banana is a severe disease affecting banana plantations, and is ranked among the top ten most important fungal diseases (Dean et al., 2012).The physiological races of F. oxysporum f. sp.cubense were unable to infect resistant banana cultivars until the 1990s, but the new race (Tropical Race 4) overcame this resistance in 1992 in Southeast Asia, and rapidly spread across banana producing areas.FocTR4 has since been reported in more than 20 countries (Bregard et al., 2022).The present study detected FocTR4 in eight of 117 plantations ( 6   peratures).This is probably why the disease is more prevalent in other countries.Karangwa et al. (2016) assessed the distribution and incidence of Fusarium wilt in Burundi, the Democratic Republic of Congo, Rwanda, and Tanzania.They reported that 54.1% of the banana plantations had disease incidences greater than 40%, and the greatest incidence (63.6%) was in Tanzania.Zheng et al. (2018) surveyed banana fields in Laos, Myanmar, Vietnam, and Yunnan Province of China, to determine presence of FocTR4.Fusarium oxysporum-like isolates were recovered from symptomatic plants from 25 banana fields, and 81.3% of these were identified as FocTR4.In a survey by Thi et al. (2022) in Vietnam, Fusarium wilt associated with Foc-TR4 was not widespread in Vietnam.Using molecular tools, these authors analyzed 19 Fusarium isolates from three different geographical regions, and found that only 10% were FocTR4.
In the present study, Fusarium isolates were obtained from banana roots, rhizomes, and internal pseudostem tissues, and this was the most frequently isolated genus (96% of isolates).In addition to Fusarium, soilborne phytopathogenic fungi and fungus-like organisms (including Macrophomina, Pythium, Phytophthora, and Rhizoctonia) associated with root and rhizome rot, and common endophytes of bananas (Alternaria, Aspergillus, Cladosporium, Epicoccum, Nigrospora, Penicillium, Trichoderma), were also isolated from these tissues.More than 100 Fusarium isolates were obtained, from which 36 that were morphologically similar to Fusarium oxysporum were selected and taken to the next stages of identification.Phylogenetic analysis showed that nine of these isolates were in the F. fujikuroi species complex, 25 were in F. oxysporum, and two in the F. solani species complex.Among these complexes, four different Fusarium species (F.musae, F. oxysporum, F. sacchari, and F. solani) were identified.Maryani et al. (2019a) obtained many Fusarium isolates from 34 geographically and environmentally different locations in Indonesia, and identified 200 of them using detailed phylogenetic analyses.The pathogen community included 14 Fusarium species within four species complexes, and 180 isolates were within F. oxysporum species complex (FOSC).Ujat et al. (2021) studied Fusarium species diversity in 17 regions of Malaysia, and found that most of the 38 isolates (86.8%) obtained were in the FOSC.Within this complex, F. oxysporum f. sp.cubense was the most frequently isolated species (71.1%), followed by F. oxysporum (10.5%) and F. grosmicheli (5.3%).Similarly, Karangwa et al., (2018) discriminated Fusarium isolates obtained from East and Central African countries using phylogenetic analysis and VCG tests, and identified F. sacchari and F. semitectum as well as Fusarium oxysporum f. sp.cubense.Although some of these F. oxysporum isolates were confirmed to be pathogenic to banana seedlings, these isolates could not be discriminated using VCG tests.Czislowski et al. (2021) obtained 105 isolates from plantations in Australia, from symptomatic and asymptomatic banana plants, recovering fungi belonging to the F. fujikuroi and F. oxysporum species complexes more frequently than those of F. solani and F. incarnatum-equiseti complexes.Fusarium Fusarium oxysporum solani and F. fujikuroi were found to predominate in the other species complexes.Results from these studies indicate that Fusarium wilt may have different prevalence in different countries, and that banana decline can be caused by non-FocTR4 species.
The TEF 1-α gene was amplified by PCR and sequenced in the present study to make robust distinction between morphologically similar Fusarium isolates.While this approach identified species within species complexes, it was insufficient to distinguish between formae speciales and physiological races within the F. oxysporum species complex.To make robust phylogenetic discrimination between Fusarium species, O 'Donnell et al. (2022) suggested that the RPB1 or RPB2 genes should be studied together with the TEF 1-α gene, but noted that TEF 1-α sequencing results would also be valuable in cases of limited resources.Ujat et al. (2021) performed phylogenetic differentiation of Fusarium isolates in bananas by sequencing the TEF or Histone-H3 genes, and suggested that working with the TEF 1-α gene revealed more consistent results than those from Histone-H3.Maryani et al. (2019a) carried out phylogenetic analyses by combining RPB1, RPB2, and TEF 1-α genes, and made detailed identification of Fusarium species complexes from bananas and the species in these complexes.Greatest phylogenetic support was obtained in their study by combining RPB1 and TEF 1 genes.Phylogenetic analyses using the sequences of gene regions proposed by Maryani et al. (2019a) could be used to identify F. oxysporum f. sp.cubense, and assess relationships among isolates.However, since the presence of FocTR4 was to be investigated in the present study by other methods (FocTR4-specific primers and a commercial identification kit), phylogenetic analyses with only the TEF 1-α gene was used as a first stage to confirm sequencing results.
The FocTR4-specific primers (FocTR4F/FocTR4R) identified eight out of the 36 selected isolates, and results were validated with a commercially available diagnostic real-time PCR kit (Clear Detections™) designed for Foc-TR4 detection.These two detection methods were used to detect first incursions of the pathogen in Colombia (in 2020) and Peru (in 2022).García-Bastidas et al. (2020) first tested the Fusarium isolates with FocTR4-specific primers to detect the presence of FocTR4 in banana plantations in Colombia.They also confirmed the presence of FocTR4 using the Clear Detection Kit™, loopmediated isothermal amplification, and whole genome analyses.Acuna et al. (2022) examined Fusarium isolates from a suspected FocTR4 infested banana plantation in Querecotillo, Peru, and detected this race in that country by conventional PCR (Dita et al., 2010;Li et al., 2015) and qPCR tests using the Clear Detection Kit™.The studies cited above, which have used various molecular identification methods, indicate that the FocTR4 can be accurately identified based on resources available in most countries.
Regarding pathogenicity, except for F. solani, the Fusarium species included in the pathogenicity tests of the present study (F.oxysporum, F. oxysporum f. sp.cubense, and F. sacchari) caused mild to severe necroses in the rhizomes of inoculated plants 40 d after inoculation.Discolouration in rhizome cores confirmed the pathogenicity of Fusarium isolates identified as FocTR4 and its association with wilt, while the other isolates were associated with root and collar rot.This study is the first to report F. oxysporum and F. sacchari causing root necrosis, collar and rhizome rots in Türkiye.Maryani et al. (2019b) examined Fusarium diversity in banana plants in small holder fields in Indonesia, and 90% of the Fusarium species isolated from pseudostems were in the F. oxysporum species complex, but members of the F. incarnatum-equiseti, F. fujikuroi and F. sambucinum species complexes were also recovered.In their pathogenicity tests, none of the Indonesian Fusarium species belonging to these species complexes (including F. sacchari) were found to be associated with Fusarium wilt in 'Cavendish' banana plants.The present study results agree with the pathogenicity results of Maryani et al. (2019b), but the present study F. sacchari isolates caused root collar and rhizome rots.Therefore, F. sacchari should not be overlooked in banana plantations.In addition, Cui et al. (2021) highlighted horizontal gene transfer between Fusarium species, indicating that the potential of F. sacchari to cause wilt in bananas should be carefully monitored.
Fusarium oxysporum isolates caused severe symptoms, although not as severe as those caused by FocTR4 isolates.Rhizome necroses started from outside and progressed towards the plant cores, suggesting that F. oxysporum isolates also cause root and root collar rot, but not wilt.Fusarium oxysporum is an important species, with numerous formae speciales and physiological races, now associated with wilt, root/crown rot, damping-off, head blight, and seed/fruit rots in many plants (Edel-Hermann and Lecomte, 2019).The host specificity of F. oxysporum, and the fact that some isolates have endophytic or pathogenic characteristics, indicate that the fungus has a complex genetic diversity (Lombard et al., 2019).Alteration of pathogenic characteristics by horizontal gene transfer between isolates has also been reported in F. oxysporum (Vlaardingerbroek et al., 2016).In pathogenicity tests by Wu et al., (2019), using the 'Cavendish' (AAA) cultivar, only FocTR4 isolates induced rhizome pith necroses, whereas this symptom was not observed in plants inoculated with Foc1 isolates.
The present study has revealed that Fusarium wilt caused by FocTR4 was present but had low prevalence (6.8%) in Turkish banana plantations.Since this race was detected in bananas in closed plastic protected greenhouses, legal and technical requirements have been fulfilled to eradicate the disease in these areas.In addition to wilt caused by FocTR4, some F. oxysporum and F. sacchari isolates used in this study were found to cause root and collar rots, and, eventually, death of banana seedlings.This is the first report of F. sacchari associated with root and collar rot of banana in Türkiye.More research is required to enable rapid and accurate pathogen detection, and to restrain the re-emergence and spread of these diseases in this country.

Figure 1 .
Figure 1.The location of surveyed banana plantations in the Mediterranean Region of Türkiye.

Figure 2 .
Figure 2. A protected banana greenhouse with wilt symptomatic plants, and vascular necrosis in an affected pseudostem.
.8%) in Türkiye, and all eight were in protected plastic greenhouses.No plants showing typical disease symptoms were found in open field plantations, and the pathogen was not detected in suspected plants.The limited detection of the pathogen in Türkiye is probably because bananas are mostly grown in protected greenhouses, with limited access and under drip irrigation systems, limiting the spread and proliferation of the pathogen.Growing conditions in other banana-growing countries are different, with most banana plantations located in open fields exposed to tropical climates (high precipitation and warm tem-

Figure 7 .
Figure7.Mean disease severity percentages in corm tissues of 'Grand Naine' banana seedlings (inoculated at the 4 to 6 leaf stage) caused by the Fusarium oxysporum f. sp.cubense TR4, F. sacchari and F. oxysporum isolates in greenhouse conditions.

Table 1 .
PCR conditions used in this study to amplify partial genomic DNA of Fusarium isolates.

Table 2 .
GenBank accession numbers of partial sequence of TEF 1-α of reference species used in the phylogenetic analyses.
): (∑ (number of plants in a disease scale category × disease scale category) / (total number of plants × maximum disease scale category)) ×100).Eight plants (one plant per pot and four replicates with two plants per replicate) were inoculated with each Fusarium isolate.Inoculation control plants were treated with sterile distilled water.Pathogenicity was confirmed by re-isolating inoculated isolates from roots and necrotic internal tissues of plants.The pathogenicity tests were each repeated once.
Figure 3. Maximum likelihood analysis of Fusarium species isolates, based on TEF-1α gene sequences.Numbers above the branch nodes represent bootstrap values from 1,000 replications.The sequence of the TEF-1α gene from F. zealandicum isolate CBS 111.93 was the outgroup used to root the tree.