Dry rot caused by Fusarium species is an important soil borne potato disease known worldwide by its serious post-harvest tuber rotting and seed piece decay after planting. This disease can reduce crop establishment by affecting the developing potato sprouts and causing crop losses estimated to up to 25% while more than 60% of tubers can be infected during storage [1]. Post-harvest tuber losses can be as high as 28% in China and 88% were mainly attributed to dry rot disease [2]. A recent study showed that in Great Britain crop losses attributed to this disease have been estimated to about 95% [3].

Fusarium dry rot (FDR) initial symptoms appear on tuber at wound sites as shallow small brown lesions after approximately one month of storage. The lesions enlarge in all directions and the periderm eventually sinks and may wrinkle in concentric rings as the underlying dead tissue desiccates [4]. Cavities underneath the rotted area are usually lined with Fusarium mycelia and spores of various colors with abundant white or carmine-colored mycelium. Fully rotted tubers become shriveled and mummified [5].

FDR is particularly prominent in Tunisia resulting in partial or almost complete loss of stored potatoes occurring especially under traditional storage conditions [6]. The past decay has been one of rapid change in the potato industry with the widespread use of refrigerated storage [3]. Although these losses can be greatly reduced by storage at low temperatures (1-5°C), dry rot is still considered a serious threat and causes important crop losses because open field traditional storage were the most practiced by Tunisian farmers.

Thirteen Fusarium species were reported to be involved in potato dry rot disease worldwide [7]. In Tunisia, F. sambucinum, F. solani, F. oxysporum, and F. graminearum were the most predominant. They were mostly isolated in combinations of two or more species [5,8-9]. Disease incidence appears to be affected by soil type, potato cultivars, Fusarium species and local climate conditions such as temperature. In fact, Fusarium species exhibited variable degrees of aggressiveness depending on storage temperatures. They have both thermal picks of aggressiveness one at lower (10-15°C) and one at higher (30-35°C) temperatures depending on the fungal complex involved in disease development [10]. Due to their large adaptive potential to temperature variation, Fusarium species are characterized by their co-occurrence within the same potato tuber and most probably by the variation of their relative dominance in refrigerated and non-refrigerated stores.

Microbial ecologists have shown that different types of interactions can occur among species, depending on biotic and abiotic factors [11]. Positive correlations can result either from direct synergy between species on the same infection site, or from indirect associations facilitated by micro-climatic conditions conducive to several species. Many reports have shown the importance of interspecific interactions between Fusarium species on many host plants such as pea [12], maize [13] and especially wheat [14] but few data were available on potato.

The most effective and environmentally approach to control FDR is the utilization of resistant potato cultivars but only cursory germplasm evaluation for resistance to this disease has been reported in literature [15,16]. In Tunisia, several reports have focused on the predominant species involved in FDR disease, the response of the local potato cultivars to the four species and the effect of temperature variation on their relative aggressiveness These screenings revealed the absence of potato cultivar combining resistance to the whole fungal disease complex and only some cultivars exhibited reduced susceptibility to at least two Fusarium species [9,17]. For a sustainable behavior of potato tubers towards the four Fusarium species involved in disease development, the type of the interaction between F. sambucinum, F. solani, F. graminearum, and F. oxysporum f. sp. tuberosi should be more elucidated on tubers based on standardized inoculation methods. In addition, environmental conditions, especially temperature, required for infection and subsequent tissue colonization may depend upon Fusarium species used for tuber inoculation. Thus, this abiotic factor i.e., temperature should be considered during qualifications of interactions between Fusarium species and breeding for multiple resistances.

In Tunisia, interactions between Fusarium species and some potato pathogens have been observed under natural conditions. In fact, F. graminearum, Rhizoctonia solani, Colletotrichum coccodes, Pythium spp. and Verticillium spp. were frequently isolated from potato plants exhibiting early dying syndrome [18-20]. Daami-Remadi et al. [21] also reported synergistic interactions occurring between V. dahliae, F. oxysporum f. sp. tuberosi and Meloidogyne javanica and leading to reduced plant growth and increase in vascular wilt severity, galling index, egg masses number and female fecundity.

Nevertheless, regarding FDR pathosystem, the relationships between Fusarium species has not been explored in detail. Therefore, this pathogen-cultivar pathological investigation may lead to a better understanding of how interactions between the four Fusarium species may affect disease incidence and severity. By understanding the interspecific interaction within the FDR complex, the obtained information will be useful for reliable disease predictions and management. Thus, the current study was set up to mimic natural infections and the overall objective is to (i) clarify the type of interaction between the four Fusarium species and assess its impact on disease development and severity based on single and mixed inoculations and (ii) determine their relative aggressiveness under two different temperatures and depending on the plant material used (i.e., potato cultivars).

Fungal inoculum

Fusarium spp. isolates were obtained from dry rotted tubers and from partially or totally wilted plants of different potato cultivars. These isolates belonged to a Fusarium complex composed of F. solani, F. graminearum, F. sambucinum, and F. oxysporum f. sp. tuberosi. Fusarium species were identified based on macromorphological, micromorphological, pathological characteristics and molecular tools based on sequencing of ITS region [22-24]. These Fusarium isolates were cultured on Potato Dextrose Agar (PDA) medium supplemented with 300 mg/l of streptomycin sulphate (Pharmadrug Production Gmbh, Hamburg, Germany). Their virulence was maintained by bimonthly inoculation of freshly wounded and apparently healthy tubers and re-isolation on PDA plates. For their preservation up to 12 months, monoconidial cultures were maintained at -20°C in a 20% glycerol solution.

Inoculum was composed of a single Fusarium species or a mixture of two, three or four species selected beforehand based on their aggressiveness (Mejdoub-Trabelsi, unpublished data). Spore suspensions were prepared by culturing isolates 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 final conidial concentration in the filtrates was adjusted to 10⁷ conidia /ml using a Malassez cytometer. An equal volume (100 μl) of inoculum was used for tuber inoculation.

Potato cultivars

Potato (Solanum tuberosum L.) tubers without physical injuries or visible infection were chosen for the bioassay. They belonged to five cultivars namely Atlas, Mondial, Nicola, Oceania, and Spunta. These cultivars were subscribed in the list A of the Tunisian varietal assortment and were kindly provided by the Technical Center of Potato and Artichoke of Tunisia. They were stored for two months in darkness at 6°C and brought to room temperature three hours before use. Prior to inoculation, tubers were superficially disinfected with a 10% sodium hypochlorite solution (Aiglol Production, Zaouiet Sousse, Tunisia) during 5 min then rinsed with sterile distilled water and air dried until use.

Inoculation and incubation

Before inoculation, tubers were wounded by removing a tuber plug (6 mm in diameter and depth) with a sterile cork-borer. These wounds were challenged with 100 μl of a single or mixed conidial suspension. For mixed inoculations, equal volumes of each Fusarium species suspension were mixed to obtain three types of mixtures (binary, ternary or quaternary) ready for tuber infection. Inoculation treatments were composed of combinations of two (six fungal treatments I,. C2-1, C2-2, C2-3, C2-4, C2-5, and C2-6), three (four treatments C3-1, C3-2, C3-3, and C3-4) or four Fusarium species (Treatment C4). Individual inocula consisted of four single treatments composed of each Fusarium species (Fusarium solani C1-1, F. sambucinum C1-2, F. oxysporum C1- 3, and F. graminearum C1-4).

The inoculated tubers (two replicates of five tubers per elementary treatment) were placed in plastic bags to maintain a high humidity and then incubated for three weeks at two different temperatures (20 or 30°C). It should be mentioned that at 30°C, the difference in aggressiveness of Fusarium species is more evident whereas at temperatures inferior or equal to 20°C only two groups can be distinguished Daami-Remadi et al. [10]. Furthermore, Mejdoub-Trabelsi et al. [9] demonstrated that when dry rot was assessed depending on temperature effect, F. sambucinum and F. graminearum developed the most severe symptoms at 15, 20 and 25°C whilst at 30°C, F. solani caused the severest rot.

After the incubation period, tubers were cut along the longitudinal axis across the inoculation sites. For disease assessment, symptomatic lesions were measured both externally and internally.

For the external evaluation, the two perpendicular diameters of the lesion were recorded and the mean diameter was calculated for each site of inoculation by using the following formula: Mean lesion diameter (mm)=(d1+d2) /2 where d1 and d2 two perpendicular diameters.

Dry rot severity was also estimated internally through the extent of the induced decay i.e., maximal width (w) and depth (d) of the rotted tissue. The pathogen penetration into tubers was calculated based on Lapwood et al. [25] formula as follow:Penetration (mm)=(w/2 + (d- 6))/2

Potato cultivars were then ranked for their susceptibility to different Fusarium treatments based on the following below scale:

•Tolerant: Lesion diameter ≤ 11 mm;

• Moderately susceptible: 11 mm

• Highly susceptible: Lesion diameter ≥ 15 mm.

Inoculation treatments were ranked for their relative aggressiveness based on the following scale:

• Less aggressive (LA): Lesion diameter ≤ 11 mm

• Moderately aggressive (MA): 11 mm

• Highly aggressive (HA): Lesion diameter ≥ 14 mm1

Statistical analyses

A factorial analysis of variance (ANOVA) was performed to determine the significance of the main factors and their interactions using a completely randomized factorial design with three factors i.e., potato cultivars, fungal treatments (tubers inoculated with single or mixed inoculum and the non-inoculated tubers) and temperatures of storage. Mean separations were performed by Duncan’s multiple range test (at P < 0.05).

Inoculated tubers of all cultivars exhibited typical external dry rot lesions expressed as brown to black flecks on the tuber surface (Figure 1). Wrinkled concentric rings appeared around the inoculation site. When cut open, infected tubers also showed light to dark brown or black rot and internal cavities containing molds within rotted tissues whereas non-inoculated control tubers remained symptomless. The intensity of these symptoms varied depending on cultivars used, inoculation treatments and temperatures of incubation tested.

Figure 1: Typical external and internal symptoms of dry rot occasionned by different mixtures of Fusarium spp. on potato tubers, noted after 21 days of incubation.

Effect of single or mixed inoculation with Fusarium species on dry rot lesion diameter

The mean diameter of dry rot lesion, noted 21 days post-inoculation, varied significantly (at P ≤ 0.05) depending on cultivars, inoculation treatments and temperatures of storage tested and their interactions. Therefore, differences between single and mixed inoculation treatments were performed for each cultivar and at each temperature (Figure 2). Overall, disease score as estimated by lesion mean diameter shows that cultivar response to Fusarium species inoculated singly or in combination varied upon the temperature used for tuber storage. In fact, for cv. Spunta stored at 20°C, the highest lesion diameter (> 15 mm) recorded 21 days-post-inoculation was noted on tubers challenged with C1-2, C2-4, and C3-3 inoculation treatments. However, when stored at 30°C, the severest dry rot lesions were observed on tubers inoculated with C4 and C3-1 treatments (composed of four and three Fusarium species, respectively). For cv. Oceania, C1-2, C2-6, C2-1, and C2-4 were found to be the most aggressive inoculation treatments at 20°C whereas at 30°C, C2-1, C2-4, C4, and C2-6 induced the highest lesion diameter. Inoculated to tubers belonging to cv. Mondial and incubated at 20°C, the mixed inoculation treatment C3-3 caused the most severe dry rot lesions (diameter of about 17 mm). However, when stored at 30°C, 12 out of the 16 inoculation treatments tested including single and mixed inoculum (namely C1-4, C3-4, C2-4, C2-3, C2-1, C4, C2-5, C1-1, C1-2, C2-2, C3-1, and C3-2) induced a significantly similar dry rot severity where the lesion diameter, noted after 21 days post-inoculation, ranged from 12.5 to 15 mm. As shown in Figure 2, cv. Atlas tubers stored at 20°C and challenged with the different inoculation treatments exhibited dry rot lesions varying in intensity (diameter 13-14.5 mm) where the most severe ones were incited by some binary (C2-1, C2-3, and C2-6) or ternary (C3-1 and C3-4) Fusarium mixtures. However, at 30°C, three single infections (C1-2, C1-3, and C1-4), four binary (C2-1, C2-3, C2-4, and C2-6), four ternary (C3-1, C3-2, C3-3, and C3- 4) and one quaternary inoculation treatments induced significantly similar dry rot lesions (diameter ranging between 10.5 and 13.5 mm). Tested on cv. Nicola tubers and incubated at 20°C, the inoculation treatments C2-1, C1-2, C2-4, C3-1, and C4 led to the development of the most severe lesions (diameter 15-17 mm). However, when stored at 30°C, the binary mixtures C2-1, C2-2 and C2-3 were found to be the most aggressive (diameter ranging from 15 to 17 mm). It should be highlighted that the treatment C2-3, ranked among the least aggressive treatments on cv. Nicola when incubated at 20°C, moved to the most aggressive group when incubated at 30°C.

Figure 2: Lesion diameter of dry rot incited by single or combined inoculation treatments with four Fusarium species tested on five potato cultivars and noted 21 days post-incubation at 20 and 30°C. Bars affected by the same letter are not significantly different according to Duncan's multiple range tests at P ≤ 0.05.

Data shown in Figure 2 also revealed that the response of the potato cultivars to the inoculation treatments tested varied depending on temperature of incubation. Moreover, the ranking of single and combined treatments, based on their relative aggressiveness, varied significantly depending on the plant material used. By examining pooled data for all cultivar × temperature combinations tested, it can be noted that some inoculation treatments were ranked within the most aggressive group at many cases. In fact, at 20°C, treatments C1-2, C2-1 and C2-4 exhibited the highest aggressiveness on 3 out of the 5 cultivars tested (cvs. Spunta, Oceania and Nicola for C1-2 and C2-4 and cvs. Oceania, Nicola, Atlas for C2-1). The combined treatments C2-6, C3-1, and C3-3 caused the severest dry rot lesions on 2 cultivars (cvs. Nicola and Oceania for C2-6 and C3-1 and cvs. Spunta, Mondial for C3-3) whereas C2-3, C3-4 and C4 induced the highest dry rot severity on only one cultivar (Atlas for C2-3 and C3-4 and Nicola for C4). However, at 30°C, C2-1 and C4 were ranked within the group of most aggressive treatments on 4 cultivars out the 5 tested (cvs. Spunta, Oceania, Mondial and Nicola for C2-1 and cvs. Spunta, Oceania, Mondial and Atlas for C4). Inoculation treatments C2-3, C2-4, and C3-1 were classified within this group on 3 cultivars (cvs. Mondial, Atlas and Nicola for C2-3, cvs. Mondial, Atlas and Oceania for C2-4 and cvs. Spunta, Atlas, Oceania for C3-1) whereas C1-2, C2-2, C2-6 and C3-4 single and mixed inoculums exhibited their highest aggressiveness on 2 cultivars (cvs. Mondial and Spunta for C1-2, cvs. Oceania and Nicola for C2-2, cvs. Oceania and Mondial for C2-6, and cvs. Atlas and Mondial for C3-4). C1-4 and C3-3 treatments showed their highest aggressiveness on only one cultivar (Mondial for C1-4 and Oceania for C3-3). Moreover, some single or mixed inoculation treatments (such as C1-1, C2-5 and C3-2) led to reduced dry rot severity for the majority of cultivar × temperatures combinations tested whereas other single treatments exhibited comparable aggressiveness as combined ones under given cultivar and temperature conditions as detailed above.

Effect of single or mixed inoculation with Fusarium species on penetration

The level of tuber decay estimated based on mean pathogen penetration depended on cultivars, fungal treatments used for inoculation and temperatures of incubation as a significant interaction was recorded between the three fixed factors. The ANOVA showed the effect of single factors and their interactions on mean penetration.

Results shown in Figure 3 indicated the mean penetration incited by the different inoculation treatments, recorded after 21 days of incubation, depending on potato cultivars used and temperatures of storage. For cv. Spunta tubers incubated at 20°C, the most aggressive treatments were C1-2, C2-4 and C2-6 where the mean penetration ranged between 10 and 12 mm. However, when stored at 30°C, the ranking of the inoculation treatments, based on the severity of the tuber internal decay, has changed. In fact, the most aggressive treatments at 30°C were found to be C4, C1-1, C3-1 and C2-1. Thus, some single treatments such as C1-1 exhibited significantly similar aggressiveness on cv. Spunta tubers as some binary (C2-1), ternary (C3-1) and quaternary (C4) treatments. For cv. Oceania stored at 20°C, the highest mean penetration (8-10 mm) was incited by C2-4, C1-2, C2-1, C3-3, and C3-4 inoculation treatments whereas at 30°C, the most severe internal tuber decay was noted on tubers challenged with C2-1, C4, C3-1, C2-5, C2-4, C2-2, and C2-6 mixed inoculums. It should be highlighted that some binary treatments (C2-1 and C2-4) were ranked within the most aggressive inoculum at both temperatures tested while C2-5 showing the least penetration at 20°C moved to the most aggressive treatment when incubated at 30°C. For tubers belonging to cv. Mondial and stored at 20°C, the severest internal tuber decay was incited by C3- 3, C2-4, C1-2, C4, C1-4 and C2-1 inoculation treatments. However, at 30°C, 10 out of the 15 treatments tested exhibited significantly comparable mean penetration. These most aggressive treatments are composed of single (C1-1, C1-2, and C1-4) inoculum and some binary (C2-1, C2-2, C2-3, C2-4, and C2-5), ternary (C3-4) and quaternary (C4) mixed infections. Data shown in Figure 3 also indicated that, at 20°C, the most severe internal decay was noted on cv. Atlas challenged with C3-4 and C2-3 mixed inoculum while at 30°C, the significantly highest penetration was incited by C2-1, C3-4, C3-1, C3-2, C4, C2- 4, and C1-2 treatments. It should be mentioned that the combined inoculum C3-4 was ranked among the most aggressive treatments on this cultivar and at both temperatures tested whereas C2-3 treatment showed reduced aggressiveness, as compared to 20°C, when incubated at 30°C. It is also important to note that the ternary combination C3-2, classified among the most aggressive treatments at 30°C, has induced surprisingly the least internal tuber decay when incubated at 20°C. Furthermore, cv. Nicola data presented in Figure 3 also indicated that some single inoculums (C1-2), and some binary (C2-1 and C2- 4), ternary (C3-1) and quaternary (C4) mixed infections led to the development of the most severe internal tuber decay as compared to the other inoculation treatments tested. However, at 30°C, the highest penetration was noted on tubers challenged with C2-1, C2-2 and C2-3 binary inoculum. It should be highlighted that C2-1 was ranked within the most aggressive group of treatments on this cultivar and under both temperatures of storage tested. However, some treatments, as is the case of C1-2 showed reduced aggressiveness on cv. Nicola when incubated at 30°C as compared to 20°C. Furthermore, C2-2 and C2-3 treatments classified within the most aggressive ones at 30°C showed reduced mean penetration (< 5 mm) when incubated at 20°C.

Figure 3: Mean penetration of dry rot incited by single or combined inoculation treatments with four Fusarium species tested on five potato cultivars and noted 21 days post-incubation at 20 and 30°C. Bars affected by the same letter are not significantly different according to Duncan's multiple range test at P ≤ 0.05.

Data shown in Figure 3 also indicated variable reaction of the potato cultivars to the different inoculums tested varied depending on temperature of incubation. Furthermore, the ranking of single and combined Fusarium mixtures, based on their relative aggressiveness, depended significantly upon the plant material used. Pooled data for all cultivar × temperature combinations tested revealed that some tested treatments were classified among the most aggressive group at many cases. In fact, at 20°C and based on mean penetration records, treatments C1-2 and C2-4 exhibited the severest internal tuber decay on 4 (Spunta, Oceania, Nicola and Mondial) out of the 5 cultivars tested. The combined treatment C2-1 led to the highest penetration on 3 cultivars (Nicola, Oceania and Spunta); C3-3, C3-4 and C4 incited the most severe internal dry rot symptoms on 2 cultivars (cvs. Oceania and Mondial for C3-3, cvs. Oceania and Atlas for C3-4, and cvs. Oceania and Spunta for C4) whereas C1-4, C2-3, C2-6, and C3-1 caused the most important tuber decay on only one cultivar. However, at 30°C, C2-1 belonged to the most aggressive group on all the 5 cultivars tested. The treatment C4 was classified within this group on 4 cultivars (Spunta, Oceania, Mondial and Atlas) whereas C2-4 and C3-1 exhibited the severest penetration on 3 cultivars (cvs. Oceania, Atlas and Mondial for C2-4 and cvs. Oceania, Atlas and Spunta for C3-1). C1-1, C1-2, C2-2, C2-3, C2-5, and C3-4 showed their highest aggressiveness on 2 cultivars (Mondial and Atlas for C1-2 and C3-4, Mondial and Spunta for C1-1, Mondial and Nicola for C2-3, Mondial and Oceania for C2-5, Nicola and Oceania for C2-2) whilst C1-4 and C2-6 were able to induce severe tuber decay only on one cultivar (Mondial for C1-4 and Oceania for C2-6). It should be mentioned that some single or mixed inoculation treatments (such as C1-3, C2-3 and C2-6) led to reduced tuber internal decay for the majority of cultivar × temperatures combinations tested whereas some single treatments (such as C1-1 an C1-2) exhibited comparable aggressiveness as some combined inoculums (as is the case of C2-1 and C2-4) under given cultivar and temperature conditions.

Consequently, all inoculation treatments were ranked into aggressiveness classes based on their disease severity estimated via the lesion diameter noted on the five cultivars at both temperatures tested. It was revealed from the results (Table 1) that the mixture C2-1 composed of F. sambucinum and F. solani was found to be the most aggressive treatment and behaved as highly aggressive on 5 cultivar × temperature combinations out the 10 tested and as moderately aggressive on the 5 other remaining ones (Figure 4). This treatment was followed by two other mixed inoculation treatments namely C2-4 and C3-4. The association of F. sambucinum with F. oxysporum (i.e., C2-4) and the combination of F. sambucinum with F. solani and F. graminearum (i.e., C3-4) were highly aggressive on 4 out the 10 cultivar × temperature combinations tested and moderately aggressive on the rest of combinations. F. sambucinum combined with F. oxysporum and F. graminearum (i.e., C3-3) or associated with all Fusarium species (C4) the obtained fungal complex behaved as highly aggressive on three cultivars by temperature combinations and as moderately aggressive on the remaining ones. Also, F. sambucinum mixed with F. oxysporum and F. solani (I,. C3-1) was ranked as highly aggressive on two combinations and as moderately aggressive on the rest. Table 1 also revealed that the four Fusarium species, when considered individually, were classified as less, moderately or highly aggressive pathogens depending on the cultivar × temperature combination tested but exhibited globally reduced aggressiveness as compared to the tested binary, ternary or quaternary complexes. It should be highlighted that all mixed inoculums including F. sambucinum showed increased aggressiveness levels on the majority of cultivar × temperatures combinations.

Figure 4: Dry rot occasioned by the most aggressive mixture of F. sambucinum and F. solani which behaved as highly aggressive (A) to moderately aggressive (B) on 5 different cultivar x temperature combinations.

Table 1: Ranking of aggressiveness of single or combined inoculation treatments tested on potato tubers belonging to five cultivars and incubated at two temperatures based on lesion diameter of Fusarium dry rot. HA: Highly aggressive: Lesion diameter ≥ 14 mm; MA:Moderately aggressive: 11 mmF. gra: F. graminearum; F. oxy: F. oxysporum; F. sam: F. sambucinum; F. sol: F. solani.

Cultivar responses to inoculation treatments tested

Table 2 shows that the differential response of the potato cultivars to the different Fusarium mixtures tested depending on the temperature of storage used. In fact, cv. Spunta behaved as highly susceptible and moderately susceptible on 5 and 18 out of 30 inoculation treatment x temperature combinations tested, respectively, while it has been ranked as tolerant on the remaining ones. This cultivar was found to be highly susceptible to C1-2, C2-4, C2-6 and C3-3 treatments at 20°C and to C3-1 and C4 when stored at 30°C. It seemed to be tolerant C1-1, C1-3, C1-4, C2-2, C2-3, C2-5 and C3-2 infections when stored at 20°C. Cv. Oceania has been classified as highly susceptible and moderately susceptible on 6 and 17 out of 30 inoculation treatment x temperature combinations tested, respectively, and tolerant to the rest of combinations. This cultivar was ranked as highly susceptible to C2-1 at both temperatures tested and to C1-2, C2-4, C2-6 and C3-3 inoculation treatments when stored at 20°C. Cv. Nicola behaved as highly susceptible to C1-2 and C2-4 when stored at 20°C and to C2-1 and C2-3 when maintained at 30°C whereas it has been ranked as tolerant to C1-1, C1-3 and C2-6 infections when incubated at 30°C. Cv. Mondial was found to be highly susceptible to C1-4 and C3-3 based treatments at 30 and 20°C, respectively and moderately susceptible at all the remaining inoculation treatment x temperature combinations tested. However, cv. Atlas tolerated C1-4 and C3-2 infections at 20°C and C2-5 treatment at 30°C and behaved as moderately susceptible to all the other treatments regardless of the temperature used.

Table 2: Differential response of five potato cultivars to single and combined inoculation treatments based on lesion diameter of FDR noted at two temperatures of storage HS:Highly susceptible: Lesion diameter ≥ 15mm; MS: Moderately susceptible: 11< mm Lesion diameter < 15mm; T: Tolerant: Lesion diameter ≤ 11 mm F. sol: F. solani; F. sam: F. sambucinum:F. gra: F. graminearum; F. oxy: F. oxysporum. C1-1: F. sol; C1-2: F. sam; C1-3: F. gra; C1-4: F. oxy; C2-1: F. sam+F. sol; C2-2: F. sol+F. oxy; C2-3: F. sol+F. gra; C2-4: F. sam+F. oxy C2-5: F. oxy+F. gra; C2-6: F. sam+F. gra; C3-1: F. sam+F. sol+F. oxy; C3-2: F. sol+F. oxy+F. gra; C3-3: F. sam+F. oxy+F. gra; C3-4: F. sam+F. sol+F. gra; C4: F. sam+F. sol+F. oxy+F. gra.

Isolation frequency of Fusarium species depending on inoculation treatments tested

As shown in Table 3, the isolation frequency of Fusarium species was variable depending on single or mixed inoculum used for tuber infection and according to cultivar × temperature combination considered. In fact, for tubers challenged with single inoculum, the isolation frequency of the different Fusarium species ranged between 62.5 and 100% depending on cultivars and temperatures of storage tested. It should be mentioned that some tuber tissues pieces plated on PDA are occasionally colonized by some other fungal species such as Aspergillus spp., Trichoderma spp. and Penicilllium spp. that inhibit Fusarium development and its isolation at 100%.

Table 3: Percentage of re-isolates of each component from individual, binary, ternary and quaternary mixtures of F. sambucinum (F. sam) or F. graminearum (F. gra) or F. solani (F. sol) or F. oxysporum (F. oxy) re-isolates from five cultivars at two temperatures. The percentage refer to eight fragments with symptoms placed on PDA plates, 2 replicates for each treatment.

Table 3 also revealed that for all binary mixtures tested, the relative isolation frequency of each Fusarium species varied from 12.5 to 75% depending on cultivars and temperatures of incubation used. It should be mentioned that F. sambucinum and F. solani have been isolated at 25 to 75% from all binary mixtures including them whereas this isolation frequency ranged from 12.5 to 50% for F. graminearum and from 12.5 to 62.5% for F. oxysporum.

Concerning the ternary mixtures, the isolation frequency of the different pathogens included in the Fusarium complex tested varied depending on inoculation treatments, cultivars and temperatures used. In fact, data shown in Table 3 indicated that the isolation frequency varied from 25 to 75% for F. solani, from 12.5 to 50% for F. sambucinum, from 12.5 to 25% for F. graminearum and F. oxysporum and this for all cultivar × temperature combinations and all mixed inoculum comprising them.

For the quaternary mixture, Table 3 also showed that all Fusarium species were successfully re-isolated with a predominance of two major species (F. sambucinum and F. solani or F. sambucinum and F. oxysporum). The isolation frequency of each Fusarium species varied from 12.5 to 50% depending on cultivars and temperatures tested. Indeed, the isolation frequency varied from 12.5 to 50% for F. solani, from 25 to 50% for F. sambucinum, from 12.5 to 25% for F. oxysporum whereas F. graminearum was scarcely recovered (12.5%).

It can be also concluded that F. solani, F. sambucinum and at a lesser degree F. oxysporum were the most frequently re-isolated agents from the majority of the ternary and quaternary inoculation mixtures tested whereas F. graminearum isolation exceeded rarely 12.5%. This may reflect their competitive potential in mixture and consequently, their relative involvement in disease development and dry rot signs scored.

The evaluation of local potato cultivars for resistance to FDR at different temperatures was previously investigated in Tunisia [9,10]. However, the present work is the first detailed study, in the world, which reports a susceptibility ranking of local potato cultivars to Fusarium species using single and mixed inoculation treatments and different temperatures. Since Fusarium species frequently co-exist in the same plant or tuber, comparative susceptibility of potato cultivars to FDR assessed based on artificial inoculation with different mixtures of four species seems compulsory. Our findings highlighted the competitive potential in mixture of F. sambucinum, F. solani and F. oxysporum. Moreover, our data are in agreement with previous data demonstrating that F sambucinum was implicated in the increased incidence of potato dry rot in USA [6], Great Britain [3]; Tunisia [8-10], Turkey [26] and Iran [27].

In this paper, inoculation with mixtures of species exhibited variation in Fusarium aggressiveness between the five cultivars used which significantly differed in terms of their resistance to dry rot development induced by single or combined species. Novelty of our findings lies in the fact that cultivars have a stronger resistance to Fusarium using single inoculum for tuber inoculation. Indeed based on cultivar differences to various Fusarium species, we would expect that resistance to Fusarium mixture was less than that observed following inoculation with each species alone, and this was the case. Mixed infections with Fusarium spp. resulted in a qualitative change of some cultivars from resistant to susceptible. This was in accordance with Bukhart et al. [28] demonstrating that although there is genetic variation for resistance to FDR, additive genetic variance is lacking or minimal, therefore genetic gains will be minimal or nonexistent with mixed inoculations. The only exception is F. sambucinum which even alone it decreases the resistance of tested cultivars. This supports the findings of Mejoub-Trabelsi et al. [9] indicating the higher aggressiveness of this pathogen. Our study suggests that double or multiple resistances did not exist in the tested cultivars. This rich activity makes the ranking of cultivar’s susceptibility more difficult and instable.

This study demonstrated that in many cases, some combined inocula were more aggressive than single ones suggesting occurrence of synergistic interaction between Fusarium species. This interspecific relation, expressed by an additional effect on the severity parameters, can explain that FDR severity has been positively affected by the combinations of Fusarium spp. Overall, mixtures of Fusarium showed higher aggressiveness than single inoculation treatments. The most aggressive mixture containing F sambucinum was not surprising. For instance, F. sambucinum was always the most aggressive comparing with the other species when tested alone. In this study, its predominance with F. solani and at a lesser degree F. oxysporum can be explained by its ability to consume nutrients before the other species (competitive exploitation), at least at these temperatures. Accordingly, Waalwijk et al. [29] suggested that F. culmorum has only competitive advantage in mixtures with F. graminearum in years characterized by cooler temperatures.

Current findings suggest that F. solani is the first to benefit from colonization by F. sambucinum, followed by F. oxysporum f. sp. tuberosi. Xu et al. [30] qualified this phenomenon by synergy, which occurs when host infection by one species leads to the host becoming more vulnerable to infection by another species. Our investigation makes clear that many mixed inoculum increased FDR severity. However, synergism cannot occur in all combinations of Fusarium species because some binary and ternary mixtures as is the case of C2- 2, C2-3, C2-5, and C3-2 infections led to least disease severity records. Therefore, the additive effect does not characterize the interactions between Fusarium species in all the cases. Indeed, with the increase of the number of concomitant species, the synergistic interactions decreased while the antagonistic interaction increased [14]. This inconsistency might be due to variability in competitiveness [31] and toxin–producing capability [32] between and within pathogen species. In the same way, Xu et al. [14] demonstrated that, when wheat inoculated with single or combinations of Fusarium species under various combinations of temperature and duration of wetness, the interspecific competition led to large reductions in fungal biomass as compared to single isolate inoculations whereas mycotoxin production increased dramatically in the co-inoculations by as much as 1000 times.

The current study revealed that, among the 11 combined inoculum tested, some mixtures containing F sambucinum did not exhibit any increase in the aggressiveness level . Thus, the most likely reason for the observed positive association among species is indirect interaction facilitated by general weather conditions conducive for disease development. Indeed, synergistic interaction occurred with some Fusarium spp. more than others. Between F sambucinum and F graminearum, there were evidence of competition. This may reflect the fact that F. graminearum and F. sambucinum are more closely related than F. sambucinum and F. solani and mainly in terms of temperature and moisture requirements for infection. In previous studies realized in Tunisia, Daami-Remadi et al. [17] showed that at less than 25°C, F. sambucinum and F. graminearum were the most aggressive, while F. solani was the most aggressive at temperature equal or superior than 30°C. The competitive advantage of F. sambucinum may be related to a better growth and spore germination rates over a wide range of temperature and potato cultivars. Indeed, the occurrence of Fusarium species is known to be largely influenced by the climatic conditions such as temperature and relative humidity [33]. The survey results strongly indicate that F. sambucinum make a tuber more suitable for F. solani, F. oxysporum f. sp. tuberosi and with less importance for F. graminearum. Similarly, it was reported by Picot et al. [34] that F. verticilloides had competitive advantage over F. graminearum, when inoculated simultaneously onto maize ears. If we focused in the fact that F. sambucinum and F. graminearum were always the most aggressive compared to F. solani and F. oxysporum f. sp. tuberosi, as demonstrated in previous work of Mejdoub- Trabelsi et al. [9], the interference competition could be a true explanation.

Since cultivar’s resistance to FDR has been positively correlated with single inoculation, the mixture of species, make selection of cultivars with combined resistance extremely difficult. In the accordance with previous findings, several other authors have reported difficulty in combining high resistance to two Fusarium species in cultivars [35]. To determine the contribution of each pathogen to the visual disease severity at each mixture, the re-isolation frequency showed that F. sambucinum, F. solani and at lesser degree F. oxysporum were the most frequently re-isolated. F. sambucinum dominated over its mixture component except when added to F. solani. In fact, the percentage of re-isolation of the last pathogen was higher (75%) than F. sambucinum mainly at 30°C from tubers belonging to cvs. Spunta, Nicola and Oceania. Growing conditions could have favored the isolation of F. solani. Accordingly, Goktepe et al. [36] demonstrated that nine isolates of F. solani responded similarly to temperature variables with an optimal growth at 30°C. The large interaction effect of cultivar and Fusarium species on the degree of rotting indicated that cultivar resistance to one Fusarium species does not confer resistance to all Fusarium species. None of cultivars tested in this study was completely tolerant to all inoculation treatments tested. Only cvs. Spunta and Oceania showed lesser susceptibility to at the most four mixtures (C2-2, C2-3, C2-5 and C3-2 for cv. Spunta and C2-2, C2-5 and C3-2 for cv. Oceania). Since none of these mixtures contained F. sambucinum, our results confirmed the higher aggressiveness of this species. This was in one part, in accordance with what was previously demonstrated in the assessment of local cultivars using single species [9]; none of the cultivars tested was completely resistant to all Fusarium species and only some of them showed lesser susceptibility to at the most one species. This is the case of cvs. Spunta, and Nicola, the most cultivated in Tunisia, which tolerated at least one species of Fusarium: F. oxysporum for the first cultivar and F. solani for the second. While cv. Mondial, which tolerated one species in a precedent investigation, presented in this study moderately susceptibility to all mixtures tested suggesting that this cultivar cannot be used for Fusarium management.

Several factors may be involved in the observed positive interactions. This interspecific relation may be outcome of complex interactions with the other factors such as varietal susceptibility, inoculum concentrations or particularly climatic conditions. In the same way, Von Der Ohe and Miedaner [32] found that aggressiveness among species and mixtures was significantly different. These differences depended mainly on the year and not on the level of host resistance. Consequently, our results emphasize the high influence of temperature on the mixture performance; otherwise, the ranking of mixtures may change with storage conditions. Thus, we can suggest that the profile of FDR disease complex, depending on cultivars tested may also be significantly affected by temperatures. Indeed, Theron and Holz [37] signaled that the different cultivars did not react uniformly to Fusarium spp. at different temperatures and they suggested that when evaluating cultivars for disease resistance or for effectiveness of some disease control measures, tests should be performed at standardized temperature. In the same way, Saremi and Burgess [38] reported the effect of temperature on the community structure of Fusarium. They found that communities of Fusarium species were significantly different at different temperatures.

It can be concluded from the current study that disease development and severity is expected to be more augmented when more than one Fusarium species were present. What remains unclear is whether this is the only factor involved for detection of resistance to FDR or whether there are others. It was thought that the mycotoxins produced by two species may be a competitive factor and that the production of secondary fungal metabolites is ecologically significant and confers increased fitness to the producing organism. Since several surveys suggested that F. solani, F. graminearum and F. sambucinum produced one or more trichothecenes, such as deoxynivalenol in North-Central United States [39], further studies are necessary in order to fully understand the diffusion of trichothecenes into potato tubers, to compare difference in trichotechenes' accumulation between resistant and susceptible cultivars and to determine the influence of temperature on the accumulation of these mycotoxins in potato tubers.

This work was done as part of research work carried out in the research unit UR13AGR09 titled Integrated Horticultural Production in the Tunisian Centre East and funded by the Ministry of Higher Education and Scientific Research of Tunisia.