Control failures of Fusarium wilt on tomatoes and resistance of cultivars to the three races of the pathogen

ABSTRACT The use of resistant cultivars is the main strategy to control Fusarium wilt on tomato, caused by Fusarium oxysporum f. sp. lycopersici . This study aimed to quantify the incidence and distribution of this disease in the region of Nova Friburgo, RJ, as well as the type of resistance of tomato cultivars to the three FOL races. Samples of healthy and wilted plants from 40 properties were evaluated for the presence of vascular discouloration. Seventeen tomato cultivars were evaluated for resistance to the three FOL races using a grading scale. Classification for resistance/susceptibility, incidence and percentage of infection of the vascular system, fresh weight accumulation and seedling length were determined. All the cultivars analysed behaved as similar to immune (SI) to race 1 of FOL and similar to immune or partial resistance to race 2. Only Aguamiel cultivar presented a SI reaction to FOL race 3. None of the cultivars presented a SI reaction to the three races. It can be concluded that failures on the control of the disease in this region can be attributed to the majority use of cultivars with incomplete resistance to the three FOL races. The seed packages also contain incomplete information about FOL resistance.


INTRODUCTION
Nova Friburgo city, a major tomato producer during summer, contributes to 13% of the production in the State of Rio de Janeiro (Emater-Rio, 2020).This production is concentrated in mountains areas of intensive management by family farmers.They have used hybrid cultivars of longlife type, reported as resistant to races 1 and 2 of Fusarium oxysporum f. sp.lycopersici (FOL) and, to a smaller extent, resistant to race 3. The use of resistant cultivars constitutes the most economical and easy to use form, besides not causing damage to the environment (Mcgovern, 2015).For this reason, it has been the main control strategy adopted by farmers in the studied region.
However, severe wilt losses and unviable areas for tomato growth are recurrent in this region.Among the wilt diseases, the Fusarium wilt, caused by FOL, is one of the most important tomato diseases worldwide (Dordevic et al., 2012;Mcgovern, 2015).Fusarium wilt can cause severe losses in protected crops and in the field (Dordevic et al., 2012).
The pathogen, which can be transmitted by seeds, causes damage to seedlings, and during the flowering and fruiting stages (Reis & Boiteux, 2007).The pathogen penetrates through the root system and colonizes the xylem vessels resulting in partial or complete disruption Control failures of Fusarium wilt on tomatoes and resistance of cultivars to the three races of the pathogen of water and nutrients, deposition of gums, thyllos, spores and mycelia, and visible discolouration of vascular tissue, leaf yellowing, wilt and death of plants (Mcgovern, 2015;Srinivas et al., 2019).
The three races of the pathogen are described by the ability to infect differential cultivars with different resistance loci, designated as immunity gene (I), introgressed from wild species (Reis et al., 2005).Among the identified R genes introgressed in S. lycopersicum are I and I-1, which confers resistance to race 1 and I-2 which confers resistance to race 2, both from S. pimpinellifolium; I-3 gene that confers resistance to race 3 and I-7, which confers resistance to races 1, 2 and 3, both from S. pennellii (Inami et al., 2012, Gonzalez-Cendales et al., 2016).In addition, the resistance gene governed by I, which confers immunity reaction to the respective races of FOL, is also reported as partial resistance resulting in delayed infection and colonization and less severe symptoms compared to a susceptible pattern.
Partial resistance genotypes often referred to tolerant, are identified by inoculation tests with isolates of races 1 and 2 of FOL and classified as highly or moderately resistant (HR and MR) (Reis et al., 2004;Carrer-Filho et al., 2016).
The predominance of resistant cultivars to races 1 and 2 of FOL is probably due to the identification of these races for more than 70 years and also to the widespread distribution in Brazil and in the world (Inami et al., 2012;Gonzalez-Cendales et al., 2016;Gonçalves et al., 2020).
As race 3 is a more recent finding, first reported in 1978 in Australia and only in 2005 in Brazil (Reis et al., 2005), the availability of resistant cultivars in Brazil is still limited.However, after the first report of race 3 in Venda Nova do Imigrante in Espírito Santo by Reis et al. (2005), several others reports have already been done in Brazil, as in Itaocara, RJ (Reis & Boiteux, 2007), Zona da Mata, MG (Gonçalves et al., 2013) and Jaguaquara, BA (Barboza et al., 2013), revealing the need to invest in resistant cultivars to this race as well.However, even using resistant cultivars to races 1 and 2 or to the three races, wilting symptoms are often reported, which leads to doubts about the nature and/ or spectrum of this resistance to FOL races in this cultivars.
Therefore, considering the report of the widespread occurrence of Fusarium wilt in Nova Friburgo, RJ, even with the use of cultivars reported as resistant to the pathogen, this work was developed.The study aimed to evaluate the incidence and distribution of the disease in the region and to identify the resistance spectrum of the cultivars used to the FOL races, type and level of this resistance.

Field harvest
Plant samples were collected in 40 tomato farms located in three association of producers in Nova Friburgo, RJ: Serra Nova, Serra Velha and Rio Grande (22º 17 '14' S latitude, -42º 32 '01'' W longitude and average annual temperature of 18.8 °C).During the surveys, the cultivars were identified, wilt occurrences and samples of plants with incipient and advanced symptoms of wilt and healthy or asymptomatic plants were harvested (table 1).

Fusarium identification and isolation
The plants were evaluated for the presence of vascular discolouration in the Laboratory of Epidemiology and Seed Pathology of UFRRJ (LabEPS).A standard procedure for fungal isolation was applied: aseptic removal of fragments from the xylem and superficial disinfestation (Baysal et al. 2009).Then, fragments of the vascular region of all plants were collected for isolation in PDA (potato-dextrose-agar) medium (Dinghra & Sinclair, 2000).The isolates were identified using the morphological characteristics and dimensions of the colonies and macroconidia, microconidia and chlamydospores with the aid of a stereoscopic and optical microscope (Booth 1977;Nelson et al. 1983).
The isolates identified as F. oxysporum were inoculated in tomato seedlings (PAB cultivar) followed by reisolation in pure culture and preserved in soil and mineral oil for further studies (Dinghra & Sinclair, 2000).
Control failures of Fusarium wilt on tomatoes and resistance of cultivars to the three races of the pathogen (race 3, 5'-GTAACCCATATTGCATGTTTCCCGGC-CGCCGCACGT-3') (MT846904 -GenBank accession number) from the UFRRJ LabEPS collection.

Experimental conditions and FOL inoculation
The experiments were performed in growth chamber The inoculation was done by cutting the tip of the seedling roots of the respective cultivars, 20 days after sowing.
For the inoculation, conidial suspension containing 1x10 6 microconidia.mL -1 of each respective isolate were used and water as a control.The roots were submerged in the conidial suspension for 5 minutes and then transplanted into tubes containing an autoclaved mixture of soil, sand, and substrate (1: 1: 1).After transplantation, 2.0 ml of the suspension was added to the respective tubes.Weekly, the plants were fertilized with 5.0 mL of Hoagland & Arnon solution (1950).For the control, distilled water was added, and the same procedures were adopted.

Analysis performed
At 18 days after inoculation, the plants were harvested and the fresh weight (g) of the shoot and the length (cm) of the stems were determined.Then, the stems were sectioned to measure the extension of vascular necrosis (EN) and grades (G) were attributed according to a scale proposed by Santos (1997), where:

Statistical analysis
The Lilliefors test for normality of errors and Cochran test for homoscedasticity of variances was carried out on all variables studied (Neter et al., 1974).Subsequentely, the data was submitted to analysis of variance (ANOVA) by the F test and the means grouped by Scott-Knott test (p < 0.05), using the SISVAR program (Ferreira, 2000).

RESULTS AND DISCUSSION
The use of 15 different cultivars were reported to be used by the farmes in the producer's regions visited.The cultivars were: Aguamiel, Alambra, Caeté, Débora Plus, Diana, Giuliana, Itaipava F 1 , Juliet F 1 , Lumi, Natália, Paron, Pizzadoro, Serato F 1 , Siluet and TSV770Cromo (Table 1).Some farmes planted more than one cultivar in the area.Others genotypes available in the LabEPS were also included in this table and in the subsequent analysis (BRS Imigrante, Carina T y , Carolina, Forty, Ivety, PAB, San Marzano and Tyler) in order to confirm or identify it's FOL resistance.
The occurrence of Fusarium wilt caused by FOL was verified in 14 farmers from the 40 farmers analysed, approximately 35% of the area.These occurrences were recorded mainly in properties with Serato F 1 .The farmers from F5, F7, F11, F13, F29 and F33 presented 10 plants in total infected with FOL, which corresponds to 42.9% from the farmers with FOL incidence.
Siluet presented FOL incidence in the farmers F6, F8, F9 and F17 and a total of 6 infected plants (28.6%), followed by Alambra with FOL incidence in two farmers F14 and F27 with 4 infected plants (14.3%).Giuliana and Juliet presented FOL incidence only in one farmer, F10 with one plant and F31 with two infected plants, respectively which represents a rate of 7.1%.For the other genotypes analysed, Fusarium wilt was not observed in theses farmers (Table 1).
Among the 17 cultivars evaluated, 14 displayed an incompatible reaction with FOL race 1, with SI reaction (I = 0, PVI = 0, G = 1), including all cultivars used in Cristiana Maia de Oliveira et al.
Carolina cultivar is informed as resistant to races 1 and 2 of FOL by its catalogue, however it behaved as susceptible to race 1 in our work (Table 1).
The relationship between the three FOL races and tomato cultivars (I-1, I-2 and I-3), is race-specific and follows the gene-to-gene theory with incompatible reaction (SI) and, therefore, independent of the isolate (Inami et al., 2012).However, the partial resistance may vary as a function of the FOL isolate aggressiveness (Carrer-Filho et al., 2016) and consistent results are obtained when more than one pathogen isolate is used (Souza et al., 2010;Akram et al., 2014).
Based on these results, it can be inferred that from the 15 cultivars marketed as resistant to race 2 of FOL, only seven (BRS Imigrante, Forty, Ivety, Lumi, Natália, Serato F 1 and TSV770Cromo) have complete resistance characterized by similar to immune (SI) (Table 2).Another six are partially resistant, two of them highly resistant (HR) (Débora Plus and Itaipava F 1 ) and four moderately resistant (Aguamiel, Alambra, Siluet and Tyler).Two, although reported as resistant, are susceptible (SU) to race 2 of FOL (Carina T y and Carolina) (Table 2).San Marzano and PAB cultivars, with no information available regarding FOL resistance, were characterized.San Marzano was considered highly susceptible to races 1 and 2, while PAB was partially resistant to FOL race 2 (Table 2).
The cultivars Alambra, Serato F 1 and Siluet are reported by the companies as resistant to FOL races 1 and 2. However, presented plants infected with FOL.This result led us to analyse two possibilities, first -these cultivars are not resistant to race 1 or 2 of FOL and second -this region presents the dissemination of FOL race 3 that can infect plants with only resistance to race 1 and 2 of FOL.
From the 15 cultivars studied and reported as resistant to race 2 (table 2), only seven displayed an incompatible reaction to race 2, including Serato F 1 cultivar.These same cultivars showed SI reaction to race 1, which confirms the simultaneous introgression of the genes I-1 and I-2 (Carrer-Filho et al., 2016).Alambra and Siluet cultivars presented incompatible reaction to race 1 (SI) and moderately resistance to race 2. Based in these results, Alambra and Siluet cultivars could be infected with race 2 or 3 of FOL, while Serato F 1 could be infected only by race 3 of FOL.This result indicates that the second possibility afore mentioned is the most presumable and the area could be contaminated with FOL race 3. Oliveira et al. (2021) 2).This result is in accordance with the survey of Mcgovern (2015) and the finding that most tomato cultivars have resistance only to races 1 and 2 of FOL.Several improved tomato cultivars present resistance to race 1 and 2 of FOL and a small number possess resistance to the three races (Reis et al., 2005;Mcgovern, 2015).
None of the 17 cultivars tested showed complete resistance, SI reaction, to the three FOL races.The only cultivar resistant to race 3, showed SI reaction to race 1 and MR only to race 2 (Table 2).From the information available in the seed packages, only in 46% of the cultivars, the resistance to race 2 was confirmed as being of immunity and, in another 46% as being of a partial nature, HR, MR or susceptible reaction (Table 2).
It is importante to consider that the resistance of cultivars to FOL is conditioned to the presence of the genes I and I-1 (race 1), I-2 (race 2), I-3 (race 3) and I-7, that promotes resistance to the three races of FOL (Gonzalez-Cendales et al., 2016;Biju et al., 2017).These genes can be introgressed simultaneously or independently.Thus, resistance will depend on the genetic configuration of tomato cultivars.
These results also reveal an emerging problem in one of the main tomato-producing regions of Brazil, the possibility of the presence of FOL race 3 and reduced supply of resistant cultivars.Another point to consider, is the Cristiana Maia de Oliveira et al. imprecision in the information contained in the company catalogues and the non-citation of the genes related to the respective reported resistances.
The plant age, methodology and environmental conditions can also affect the results of genotype characterization for partial resistance (Boix-Ruíz et al., 2015).The methodology adopted is the same used by different authors (Souza et al., 2010, Carrer-Filho et al., 2016;Gonçalves et al., 2020), as well as the initial screening and identification of SI reactive materials.However, as it is a drastic method it may hinder the identification of genotypes bearing partial resistance.The stability of this resistance must be verified in field studies and evaluated until the adult plant (Cantú et al., 2014).
In general, the inoculations using four isolates resulted in a significant reduction of the fresh weight and plant growth, even in incompatible interactions, where loss from 50% to 80% were registered compared to the non-inoculated plants.Higher losses, however, were observed in the plants of the cultivars with HS reaction -Ponderosa (60 to 90%).Differences were also observed between isolates, with higher losses caused by the FUS 2903 isolate of race 3 and lower by the isolate MMBF 02/96 of race 2 (Table 3).

(
28/25 ºC day/night, relative humidity, 70%).A completely randomized experimental design was performed with 21 cultivars.Each genotype was represented by 10 plants, seven inoculated with the FOL races (1, 2 or 3) and three as control.The experiment was repetead at least three times to verify its reproducibility.The seedlings from each cultivar were germinated in the greenhouse and inoculated at 20 days after sowing.
(1) plants without symptoms; (2) plants without symptoms of wilt or yellowing, but with vascular browning; (3) plants with vascular browning and wilt or foliar yellowing; (4) intensively wilted plants, associated with yellowing and foliar necrosis; (5) dead plants.The relative fresh weigth (RFW), was estimated by the relation between fresh weigth data of inoculated plants (FWI) and non-inoculated plants -control (FWC) where, RFW = (FWI / FWC) * 100.Relative lenght (RL) was estimated by the relation between stems lenght data of the inoculated plants (SLI) and the controls (SLC), where RL = SLI / SLC * 100.The percentage of vascular infection (PVI) was determined using the data from SL and the extent of necrosis(EN), where PVI = (EN / SL) * 100.The cultivars were classified using the grade average (G), according to model developed byReis et al. (2004), where: 1.0 = similar to immune (SI); 1.1-2.0= highly resistant (HR); 2.1-3.0 = moderately resistant (MR); 3.1-4.0= susceptible (SU); 4.1-5.0= highly susceptible (HS).The incidence of plants with disease symptoms was determined by the relation between infected plants and total of inoculated plants, considering each race of the pathogen.

Table 1 :
List of tomato cultivars, company with respective information regarding the comercial group and resistance to the races 1, 2 and 3 of Fusarium oxysporum f. sp.lycopersici (FOL).FOL incidence was obtained from the plants diagnose in loco and using differential cultivars to the races of the pathogen

Table 3 :
Relative fresh weight of shoots (RFW) and relative lengh of stems (RL) from tomato plants inoculated with four Fusarium oxysporum f. sp.lycopersici isolates, races 1, 2 and 3, compared with non-inoculated plants