EFFECT OF SINGLE AND COMBINED INOCULATIONS OF POTATO PLANTS WITH FOUR FUSARIUM SPECIES ON WILT SEVERITY, PLANT GROWTH, AND PRODUCTION

Article history Received: March 21, 2020 Revised: May 25, 2020 Accepted: August 05, 2020 Potato (Solanum tuberosum L.) Fusarium wilt is an increasingly serious disease in many potato-growing regions worldwide and notably in Tunisia. Fusarium sambucinum, F. oxysporum, F. solani, and F. graminearum are the main causal agents with F. oxysporum being the most aggressive on plants. Co-infection with this Fusarium species complex is frequently observed. Cultivar responses to different pathogen mixtures can give additional information on the relative aggressiveness of the different complexes. This study may lead to a better understanding of how interactions between the four Fusarium species may affect disease incidence and severity. Interactions between these Fusarium species was evaluated using single and combined inoculations (15 different Fusraium inocula) onto potato the two cultivars Spunta and Oceania. The tested inocula were evaluated for their effects on leaf yellowing and necrosis, vascular discoloration severity, potato growth and production. Necrosis severity noted 75 days postplanting induced by three different inocula C2-1 (F. sambucinum + F. solani), C2-2 (F. solani + F. oxysporum), and C3-3 (F. sambuinum+F. oxysporum +F. graminearum) were found to be the highly aggressive inoculation treatments. Overall, all mixed inocula including F. sambucinum showed increased aggressiveness levels. The two cultivars exhibited differential response to the different tested Fusarium mixtures and behaved in the majority as sensitive to moderately sensitive to C1-1, C14, C21, C2-2, C2-3, C2-4, C2-5, C2-6, C3-1, C3-3, C3-4 and C4 treatments. This study clearly demonstrated that Fusarium wilt of potato is expected to be more severe when more than one Fusarium species is present. The relative predominance of F. sambucinum may reflect its competitive potential in mixture and its significant involvement in potato Fusarium wilt severity.


INTRODUCTION
Potato (Solanum tuberosum L.) losses attributed to fungal diseases are becoming the major worrying constraints faced by potato growers worldwide. Among the widely encountered biotic problems, Fusarium wilt is an increasingly serious soilborne disease as the causal agents (various Fusarium species) are difficult to control due to their soilborne origin and inevitable occurrence (Munkvold, 2016). Many potato organs (tubers, roots, stems, etc) may be infected by Fusarium species leading to root and tuber rots and eventual plant wilt witling in addition to tuber contamination by mycotoxins (Pernezny et al., 2014). These pathogens are responsible for severe vascular wilts, root and crown rots in a diverse group of crop plants (Enya et al., 2007). In fact, Fusarium wilt diseases rank among the most devastating diseases, constituting a significant agricultural threat (Fisher et al., 2012). This problem persists throughout the world and is prevalent in the most potato-growing areas leading to 30-50% yield losses and decreased quality and safety of tubers intended for consumption (Ommati and Sharifi, 2008;Kerkeni et al., 2013). Fusarium wilt has been identified as one of the most yield limiting disease of potato, worldwide (Ommati et al., 2013). Fusarium oysporum is infamous for causing vascular wilt diseases in over 100 different crops (Edel-Hermann and Lecomte, 2019). In Tunisia, F. oxysporum and F. solani are the main species involved in plant wilting Ayed et al., 2006). In addition, they are frequently associated to tuber dry rot (Daami-Remadi and El Mahjoub, 2004). Infection of plants may be initiated on roots through natural openings or wounds before invading the xylem vascular tissues. This infection process leads to plant stunting, reduced growth, leaf yellowing, vascular browning, and eventual total wilting of severely infected stems . The literature on this disease is not extensive, although its occurrence in many potato producing countries (Saremi and Amiri, 2010;Xue et al., 2013). In addition, Fusarium Wilt is most often associated with infections with other bio-agressors such as the root lesion nematode and other fungal and bacterial agents responsible for the known early potato dying syndrome (Daami-Remadi et al., 2008). In fact, when multiple pathogens are present, interactions may result in additive or synergistic increases in disease incidence and severity as well as decreases in yield (Hou et al., 2020). In this context, previous studies demonstrated that Verticillium dahliae and Pectobacterium spp. can individually have significant detrimental effects on potato production, but when they are present together, their destructive potential can be significantly increased (Rowe, 1987). Another study reported that plants co-inoculated with both V. dahliae and P. carotovorum subsp. carotovorum exhibited more severe chlorosis and stem soft rot symptoms as compared to plants inoculated with either pathogen alone (Rahimian, 1984). In the same way, Dung et al. (2013) indicated that co-infection by Pectobacterium and V. dahliae can result in increased potato early dying severity.
For an efficient Fusarium wilt control, the use of resistant varieties is the most reliable approach especially in terms of preservation and respect of environment and human health. Esfahani (2005) recorded a varied response of potato varieties to Fusarium wilt caused by F. sulphureum, F. solani and F. oysporum. In Tunisia, many research efforts attempted to select a potato variety resistant to Fusarium species involved in this disease but no combined resistance was found but only some varieties displayed tolerance to two Fusarium species at most Mejdoub-Trabelsi et al., 2012). In addition, Selection pressures attributed to the host, as well as inter-and intra-specific competition drive the evolution of fungal genomes (Rampersad, 2020). Faced with this situation, the study of the interaction occurring between all Fusarium species complex is required. Obtaining a high level of resistance to the whole pathogen complex seems to be closely related to the relationship characterizing the involved Fusarium species. Their frequently observed coexistence in nature encourages us to associate them through mixed inoculations, thus imitating the concrete situation encountered under natural conditions during potato growing cycle. Our previous research (Mejdoub-Trabelsi and Ben Abdallah, 2016), more focused on Fusarium dry rot disease, has led to some very interesting and promising results where we demonstrated that disease development and severity is more severe when more than one Fusarium species is present suggesting an additive effect between them during their interaction (F. oxysorum, F. solani, F. sambucinum, and F. graminearum). However, to the best of our knowledge, the relationships between pathogen species responsible for Fusarium wilt disease have not been previously explored. Therefore, the major aims of current investigation were to (i) elucidate the type of interaction occurring between the four Fusarium species causing potato Fusarium wilt and to assess their effects on wilt development and severity based on single and mixed inoculations and (ii) determine the relative aggressiveness of the tested inocula depending on potato cultivars.

Fungal inoculum
Plant infection was performed using a multiple set of inoculum, i.e., a single Fusarium species, a mixture of two, three and four Fusarium species. Spore suspensions were prepared by culturing Fusarium spp. in Potato Dextrose Broth (PDB). After incubation for 7 days at 25 °C under continuous shaking at 150 rpm, the cultures were filtered through two layers of cheesecloth to remove mycelium and then through two layers of Whatman No. 1 filter paper. The obtained suspension was filtered and equal volumes of conidial suspensions from each Fusarium species were mixed and the final concentration of the combined inoculum was adjusted to 10 7 CFU/mL using a Malassez hemacytometer. The different types of inocula used for plant inoculation were detailed in Table 1.  , were used in this pot experiment. They were previously stored at 6 °C for two months before being used. They were superficially disinfected using a sodium hypochlorite solution diluted at 10% during 5 min, rinsed with tap water and air dried. They were maintained under ambient conditions for germination.

Plant inoculation
The mixed Fusarium species were evaluated on potato plants for their relative aggressiveness as Fusarium wilt agents compared to single inoculations. Seed tubers, showing optimal germination, were planted in individual pots containing a mixture of perlite and peat (1:3 v/v). Two weeks after emergence, potato seedlings were infected with the different Fusarium spp. inocula (applied singly or in combination) by watering each pot with 100 ml of a conidial suspension (10 7 CFU/mL). Uninoculated control plants were watered with 100 ml of sterile distilled water only (Daami-Remadi and El Mahjoub, 2004). All treated pots were placed under greenhouse conditions (18-25 °C, 14 h light) for 60 days and watered regularly enough to avoid drought stress. Ten plants were used per individual treatment. The whole experiment was repeated twice. Fusarium wilt severity was assessed, 60 days postinoculation, based on the intensity of leaf yellowing and necrosis. This parameter representing the intensity of foliar symptoms was noted using the following arbitrary 0-4 scale (Kerkeni et al., 2013) where 0 = asymptomatic leaves, 1 = Wilted leaves, 2 = Leaves showing unilateral yellowing, 3 = Leaves showing unilateral necrosis, and 4 = Dead leaves ( Figure 1). The second disease severity parameter is the index of the vascular discoloration estimated through longitudinal sections of stems and measurement of the length of this staining from the collar to the end of the stalk. This evaluation was performed per stem and the average for all stems was calculated per plant. Then, this parameter was classified according to an arbitrary scoring scale of the vascular discoloration (browning) index ranging from 0 to 4 ( Figure 2) as follows where 0: No vascular browning; 1: Browning ≤ 1 cm; 2: 1 cm < Vascular browning ≤ 3 cm; 3: 3 cm < Vascular browning ≤ 5 cm; 4: Vascular browning > 5 cm.  Other growth parameters such as the fresh weight of the aerial part and tubers were noted to evaluate the effect of simple and mixed inoculations on plant growth and production. The classification of fungal treatments (single and mixed inocula) tested on plants, as Fusarium wilt agents, was performed based on necrosis index records using the following arbitrary scale:

RESULTS
All plants inoculated with Fusarium spp. showed typical symptoms of Fusarium wilt. Thus, all fungal treatments, tested singly or in mixtures, were found to be pathogenic on potato plants. However, wilt severity varied depending on parameters noted, nature of the inoculum used for plant inoculation and cultivars tested.

Leaf necrosis severity
Leaf necrosis severity, noted 75 days post-planting, varied significantly depending on tested cultivars and fungal treatments used for plant inoculation.

Vascular browning severity
Fusarium wilt severity, evaluated based on the index of vascular browning, varied significantly depending on tested fungal treatments. Data given in Figure 4 showed that for all cv. Spunta plants, the vascular browning index did not exceed 2 where only 05 treatments out of the 15 tested led to the development of a vascular browning with an index ranging between 1.55 and 2. These treatments are composed of the tetra-specific inoculum (C4), 02 tri-specific treatments (C3-3, C3-4), the bi-specific inoculum C2-4 and the mono-specific inoculum C1-4. It is important to note that plants inoculated with C3-2 Fusarium species complex showed the lowest browning index (0.5) which was significantly comparable to the uninoculated control and which was 4 times lower than that noted on those infected with C1-4. As for cv. Oceania response to the different inoculation treatments, 05 out of the 15 tested inocula induced a vascular browning with an index ranging between 1.2 and 2. The concerned treatments were C2-5, C3-3, C4, C1-4 and C2-4. However, two trispecific (C3-1 and C3-2), four bi-specific (C2-2, C2-3, C2-6 and C2-1) and three mono-specific (C1-2, C1-3 and C1-1) inocula induced a vascular browning with a comparable severity the uninoculated control.  Table 1.
As the leaf necrosis was the sole disease severity parameter which varied at the same time depending on cultivars used and tested inoculation treatments, all inoculum types (mono, bi, tri and tetra-specific) were ranked based on their relative levels of aggressiveness on the tested cultivars ( Table 2). As for ratings of single-specific inocula as wilting agents on cv. Spunta, only C1-4 (F. oxysporum) out of the four mono-specific inocula tested was found to be highly aggressive. However, on cv. Oceania, all mono-specific inocula were moderately aggressive except C1-2 (F. sambucinum), which was not-aggressive on potato plants.
As for the ranking of of the bi-specific treatments when challenged to cv. Spunta, the six inocula were found to be highly aggressive whereas on cv. Oceania, they were in majority moderately aggressive except C2-1 (F. sambucinum + F. solani) and C2-2 (F. solani + F. oxysporum) which were shown to be highly aggressive.

Effect of simple or mixed inoculations with Fusarium spp. on potato growth and production parameters
Analysis of variance performed for the three studied parameters (weight of the aerial part, roots and tubers) revealed a highly significant interaction (at P < 0.01) between the two tested factors (cultivars and fungal treatments).

Aerial part fresh weight
The effect of mono and multi-specific inoculations with Fusarium species based on the fresh weight of the stems is shown in Figure 6. This figure shows that all single and mixed inocula tested, regardless of the varieties used, had significantly reduced the fresh weight of the aerial part by 27.51 to 59.26% compared with the uninoculated control. Figure 6. Effect of single and mixed inoculations by four Fusarium species on the aerial part fresh weight noted on two potato cultivars 75 days post-planting Bars sharing the same letter are not significantly different according to Duncan's Multiple Range Test at P ≤ 0.05. NIC: Uninoculated control, Tetra: Tetraspecific, The composition of all tested inoculation treatments is detailed in Table 1.
As for the specific effects of the tested mono and multispecific inocula based on the fresh weight of the aerial part, the inoculation of cv. Spunta with the single-specific inocula had reduced this parameter by 13.49 to 41. 25% compared to control. However, on cv. Oceania, monospecific inoculations led 3.69 to 47.55% lesser aerial weight relative to control. As for the bi-specific inocula challenged to cv. Spunta, the treatment C2-2 (F. solani + F. oxysporum) reduced the weight of the aerial part of 72.81% versus control.
The tri-specific inocula tested on cv. Spunta decreased this parameter by 36.92 to 61.81% compared to control plants whereas reduction ranged between 36.25 and 62.57% when challenged to cv. Oceania. Inoculation by the tetraspecific inoculum led to 60% decrease in Spunta versus 59.91% in Oceania.

Root fresh weight
The root fresh weight varied significantly (at P < 0.05) depending on cultivars used and fungal treatments tested. The respective effects of the different inocula on tested cultivars are presented in Figure 7.  These fungal treatments were followed by two monospecific inocula namely C1-3 (F. graminearum) and C1-4 (F. oxysporum) which lowered this parameter by 38.33 and 37.41%, respectively. The third group of relatively important inocula, based on the fresh root weight reduction compared to control, is composed of C1-2 (F. sambucinum) and C3-2 (F. solani + F. oxysporum + F. graminearum) with 32.05 and 30.37%, respectively. These fungal treatments were followed by C1-1 (F. solani) and C2-4 (F. sambucinum + F. oxysporum) leading to 23.19 and 25.54% decreases in this growth parameter, respectively. Treatments C2-5 (F. oxysporum + F. graminearum), C2-6 (F. sambucinum + F. graminearum) and C4 reduced this parameter by 5.59 to 11.18% versus control.

Tuber fresh weight
This parameter varied significantly depending on cultivars tested and fungal treatments used for plant inoculation; a highly significant interaction (at P ≤ 0.01) was recorded between these two factors.
Concerning the relative effects of single and multispecific inocula on this production parameter, Figure 8 shows that all tested treatments led to significant decreases in tuber yields in both cultivars as compared to control. In fact, following inoculation of cv. Spunta with the tested monospecific inocula, there was a decrease in the tuber fresh weight estimated at 2.36 to 50.42% depending on Fusarium species. However, for cv. Oceania, the recorded yield reductions ranged from 21.36 to 36.22%.
As for the effects of bi-specific inocula, Figure 8 shows that the tuber weight loss varied between 0.73 and 20.38% on cv. Spunta and between 24.34 and 50.41% on cv. Oceania. Tri-specific inocula applied to both cultivars led to reduced tuber weight ranging from 0.21 to 17.32% on cv. Spunta and from 18.98 to 30.27% on cv.

DISCUSSION
In the last decades, plant pathologists have become increasingly interested in the concept of ecological niche and species coexistence (Kennedy, 2010). In fact, within the same ecological environment, different mechanisms can allocate benefits to one species over another. Indeed, in the current investigation, we studied the interaction that can occur between different Fusarium species colonizing the same ecological niche where a specific interaction leads to the predominance of some species over others. Although it is clear from previous research that individual infection by F. oxysporum or F. solani can result in wilt chlorosis and premature senescence, effects of F. sambucinum and F. graminearum, as causative agents of potato Fusarium wilt, was not deeply investigated. In addition, in order to search for tolerant plant material, these most prevalent Fusarium species in Tunisia should be considered in combination as a complex for getting a more sustainable response of potato cultivars to Fusarium wilt. For these reasons, a set of eleven combinations of Fusarium species was studied for the first time in the current investigation. The results of this study revealed that plant inoculation with various mixtures of Fusariums species led to varied infection severity and cultivar responses. These findings described the involvement of an aggressive association which is the tripartite association composed of F. oxysporum, F. sambucinum and F. graminearum. It is important to note that, according to our previous investigations (Mejdoub-Trabelsi and Ben Abdallah, 2016), the mixed infections ranked was being the most aggressive on tubers (as dry rot agents) are different from those classified as aggressive on plants (as wilt agents) in the current investigation. Therefore, in seeking this multi-specific resistance, each Fusarium species should be considered separately. Moreover, presence of F. oxysporum, as a component in this association, is not strange because this pathogen has been a fixed component in all mixtures ranked as the most aggressive on plants. Focusing on the type of interactions, the association of F. sambucinum, F. graminearum and F. oxysporum showed increased aggressiveness on plants; suggesting the existence of a synergism between them.
In an attempt to explain this phenomenon, let us remember that on plants, soil factor and the ascending evolution of the species could not be neglected. In fact, F. oxysporum, F. sambucinum and F. graminearum do not have the same requirements in the soil and then in the roots and vessels of the plant (Saremi and Amiri, 2010); this could explain their aggressiveness when they have been associated and their needs do not interfere with each other. These observations lead us to focus on a positive association rather than a negative combination. In this context, Stachowicz (2001) as well as Bruno et al. (2003) suggested that the development of one species may make the environment more favorable for others; which positively affects the structure of the fungal community and increases the aggressiveness of the complex. Since reacting together on plants, competition is by far the relationship that lies between them on plants. Xu et al. (2007) by evaluating the effect of F. avenaceum, F. culmorum, F. graminearum, and F. poae as simple and mixed inoculations on mycotoxin concentrations, demonstrated that the observed interactions suggest rather the presence of competitive interactions. They recorded a reduction in fungal biomass and a more than 1000-fold increase in mycotoxin production was detected following co-inoculations. The interaction of the four Fusarium species differs from those observed on tubers (Mejdoub Trabelsi et al., 2015) where the mixed infection, attributed to F. sambucinum and F. solani, was found to be the most aggressive on tubers incubated at 20 and 30°C. Thus, F. sambucinum seems to be the point of intersection of the two most aggressive associations among all tested inocula. This species seems to colonize the tubers perfectly and probably colonize extensively the other plant tissues. Wicklow and Caroll (1981) have long reported that, in competition between fungi, theoretically occupying the same econiche, two features are often intertwined or overlapping i.e. competitive exploitation or interference competition. The first refers to the ability of a given species to consume nutrients before another present while the latter involves chemical or adaptive behavior that limits the access of competing species to resources. In addition, the presence of F. sambucinum or F. oxysporum in the most aggressive complexes does not support the hypothesis that one of them carries the competitive advantage more than the other. The idea of chemical association between these four species cannot be rejected either. This complicity between species can only be affected by factors other than types of associations such as cultivar sensitivity. Despite the importance of these results, the use of "consortium" of Fusarium species to select the most resistant genotypes to multiple species is more or less questionable according to some authors. Indeed, Reid et al. (1999) indicated that the occurrence of interspecific competition is possible and furthermore, it varies with environmental conditions, including temperature. They suggested the avoidance of complicated interactions that may occur between different species and that the use of species mixtures is not appropriate for the selection of F. graminearum and F. moniliforme resistant maize. In the current study, after detailing the different effects of single and mixed infections on disease severity and plant growth and production, we suggest variable responses of the tested consortia depending on grown cultivars and growth conditions. In fact, this type of investigations involves several factors that may complicate the interpretation of the obtained results but in this work the Fusarium species complex was monitored taking into account all the factors of the disease triangle. However, these conclusions may be more accurate by performing re-isolations from infected plants to determine which Fusarium species predominates in the complex and may be the more involved in the observed and evaluated symptoms. This complement study may be useful to determine the nature of the relation occurring between tested Fusarium species, positive or negative, in the light of suggestions (Xu et al., 2007). The current study demonstrated for the first time in the world the specific effects of various single and mixed infections with Fusarium species on wilt severity, subsequent yield loss, and cultivar response. We conclude that Fusarium wilt of potato is expected to be more severe when more than one Fusarium species is present. The relative predominance of F. sambucinum may reflect its competitive potential in mixture and its significant involvement in potato Fusarium wilt severity.
Based on the findings of this research and considering the higher positive effects of co-inoculation than individual pathogens, management of the Fusarium wilt should be carried out using more cultivars where more than one species are present. In order to improve potato production in this region, this investigation may be completed by a concomitant monitoring of the evolution of tested inocula in the soil following these recorded interactions.

ACKNOWLEDGMENTS
This work was funded by the Ministry of Higher Education and Scientific Research of Tunisia through the funding allocated to the research unit UR13AGR09-Integrated Horticultural Production in the Tunisian Centre-East, Regional Research Centre on Horticulture and Organic Agriculture of Chott-Mariem, Tunisia.