EFFECT OF NACL ON THE DEVELOPMENT OF THE OIL PALM VASCULAR WILT FUNGUS, FUSARIUM OXYSPORUM F. SP. ELAEIDIS

Oil palm Elaeis guineensis Jacq. (Arecaceae) is one of the cash crops that mainly contributes to the gross domestic production of growing countries. This crop is grown worldwide throughout the humid intertropical belt. Its production in Africa is mainly threatened by the telluric fungus Fusarium oxysporum f. sp. elaeidis, the causal agent of the oil palm vascular wilt. It has been reported that some environmental factors, such as soil and air salinity, may determine the survival, development and severity of phytopathogenic agents including Fusarium. The purpose of this study was to evaluate the effect of NaCl on growth, reproduction and pathogenic traits of this fungus by measuring mycelium daily growth, mycelium dry weight, sporulation rate, germination rate and number of infectious spores under four NaCl concentrations (0 g/L, 2.5 g/L, 5 g/L ,10 g/L) of culture medium (MM solid medium and Armstrong liquid medium). The results indicated that NaCl reduced radial daily growth and mycelium dry weight. In addition, the NaCl reduced significantly the sporulation rate and the number of infectious spores while increasing the germination rate. Overall, these results indicated the negative NaCl effect on the development and the fitness of this pathogenic fungus. Thus, NaCl inputs appeared to be a potential solution for managing Fusarium oxysporum f. sp. elaeidis in the field, if a good balance between a decrease of disease incidence and yield loss is reached.


INTRODUCTION
Oil palm, Elaeis guineensis Jacq. of the Arecaceae family is grown worldwide throughout the humid intertropical belt. It represents a major source of cash income for the growing countries (Allou et al., 2017). Among all oilseed crops, oil palm brings the highest yields per hectare (Baron et al., 2017) and provides more than one-third of the world's vegetable oil production (Jacquemard, 2012). These vegetable oils are used in food and soap manufactures including cosmetics; one percent of biodiesel comes from palm oil (FAO, 2013). However, oil palm production is subjected to fungal and bacterial attacks. Among these fungal diseases, Fusarium vascular wilt is responsible for significant damage in West and Central Africa (Flood, 2006;de Franqueville et al., 2011;Ngado et al., 2013), causing up to 70% mortality in the field. It is the main constraint for oil palm production in Africa (Ntsomboh-Ntsefong et al., 2015). This disease is caused by a telluric fungus known as Fusarium oxysporum f. sp. elaeidis (Foe) (de Franqueville et al., 2011;Gogbe et al., 2016). The pathogen penetrates the plant through the injured roots using aggressive enzymes, then goes through the epidermal barriers to move upwards along the intra-and intercellular pathways of the xylem to the pseudobulb. Accumulation of gum in the xylem vessels lead to a total obstruction of the vessel light and prevent the flow of the sap (Tengoua and Bakoumé, 2008). In worst cases, it results in more or less rapid desiccation of the affected palm-trees preceding the death (Asssohoun et al., 2016). Development of phytopathogenic fungi and their incidence on hosts are highly conditioned by environmental (Renard and Ravisé, 1986;Xu et al., 2008) and climatic factors (Rossi et al., 2001;Doohan et al., 2003). Among these, mineral soil composition and particularly salinity are known to potentially have a critical impact on phytopathogenic fungi development.
Indeed, soil and air salinity are one of the major environmental factors that may conditioned survival, development and incidence of phytopathogenic fungi (Regragui, 2005). Thus, soil salinity might be a potential way of management of fungal diseases in the field. However, opinions are divergent with regard to the effect of NaCl on phytopathogenic fungi, and especially on Fusarium species. The effect of salinity varies from one species to another, either directly or indirectly. In vitro tests were performed to evaluate the effect of NaCl on mycelial growth, sporulation and spore germination of Verticillium albo-atrum, Phytophthora capsici, Fusarium oxysporum f. sp. radicis-lycopersici (Regragui, 2005;Sanogo, 2004;Triky-Dotan et al., 2005). Regragui (2005) showed that NaCl stimulates mycelial growth, sporulation and spore germination of Verticillium alboatrum while Sanogo (2004) found a positive effect on growth parameters but a negative effect on reproduction ones with Phytophthora capsici. Some studies revealed that the salinity of soil and air affects the development of Fusarium genus species (Mecteau et al., 2008;Triky-Dotan et al., 2005). Furthermore, Triky-Dotan et al. (2005) observed that NaCl affect negatively reproduction parameters of Fusarium oxysporum f. sp. radicislycopersici; while Ragazzi and Vecchio (1992) observed that germination and germ tube length of chlamydospore of Fusarium oxysporum f. sp vasinfectum increase in substrates containing sodium chloride. Overall, these results showed that NaCl can affect the growth and reproductive parameters depending on the fungus species. Therefore, the pattern of the NaCl effect on the growth and reproductive parameters of a particular species of fungus including Foe cannot be predicted. In fact, the influence of NaCl on the development and reproduction of F. oxyporum and particularly of Foe, as well as their potential use to control the pathogen, has been poorly studied and discussed. This study aims at evaluating the influence of NaCl on (i) the growth; (ii) the reproduction ability and (iii) the infectiveness of Foe to evaluate its potential use as a method to control Foe in oil palm plantations. MATERIALS AND METHODS Isolation of Foe: The isolate of Fusarium oxysporum f. sp. elaeidis used in this study is the isolate IB7, routinely used for screening test in the laboratory, and kept in the fridge at 4°C on compost soil. It was isolated in southern Benin (Guinean zone, Atlantic Department, 6°40'0'' N and 2°15'0''E) from a stipe of an oil palm affected with fusariosis, which was incubated on the MM culture medium. This strain was then subjected to a pathogenicity test which confirmed its virulence. NaCl solutions and media preparation: MM medium was prepared using 1g di-Potassium Hydrogen Phosphate (Panreac), 0.5 g Iron Sulphate (VWR Chemicals), 1.5 g Asparagine (Merck KGaA), 1g Yeast Extract (BD), 25 g Agar agar (Kalys) and 1-liter distilled water. Armstrong medium was prepared using 20 g Glucose (VWR Chemicals), 0.4 g Magnesium Sulfate (VWR Chemicals), 1.6 g Potassium Chloride (VWR Chemicals), 1.1 g Potassium dihydrogen phosphate (VWR Chemicals), 5.9 g Calcium Nitrate (VWR Chemicals), 0.01 g Iron Chloride (VWR Chemicals), 0.01 g Manganese Sulfate (VWR Chemicals), 0.01 g Zinc Sulfate (VWR Chemicals) and 1-liter distilled water. Four NaCl (Qualikems) concentrations have been used: 0 g, 2.5 g, 5 g and 10 g per liter of culture medium, and 10 replicates were performed for each concentration. Inoculation and incubation of Foe: To assess the radial daily growth, a MM solid medium Petri dish was prepared and inoculated at the center with a small calibrated amount of compost soil containing Foe. The Petri dish was placed in the incubator at 28°C for 10 days. Then, 10 Petri dishes of MM solid medium of each NaCl concentration were prepared and inoculated at the center with one culture fragment of size 1cm X 1cm. The forty Petri dishes were placed in the incubator at 28°C for 10 days. To assess the sporulation rate, germination rate and the number of infectious spores, 5 ml of distilled water were poured into each Petri dishes and the mycelium was scraped off with a microscopic slide. The mycelium suspensions were collected in test tubes and kept in the incubator at 28°C for three days in order to enhance spore germination. To assess the mycelium dry weight, one Petri dish of MM solid medium was prepared and inoculated at the center with a small calibrated amount of compost soil containing Foe. The Petri dish was placed in the incubator at 28°C for four days. Then, one culture fragment 1cm X 1cm was collected and used to inoculate 100 ml of Armstrong medium in an Erlenmeyer flask. This was then placed in a dark culture room for 4 days incubation at 29°C and shaken moderately three times a day. Then, 40 Roux flasks (10 for each NaCl concentration) containing each 100 ml of Armstrong medium were inoculated with 2 ml of the initial inoculum. Roux flasks were then placed in a dark culture room for 10 days at 29°C and shaken moderately three times a day.
Afterwards, the culture was filtered using filter paper and transferred on test tubes previously weighted. Data collection: Colony diameter (mm) in each Petri dish was measured every day during the incubation period at 9 am to assess radial daily growth of mycelium. Average radial daily growth (i.e. computed over the whole incubation period) and radial daily growth (i.e. radial growth from day D to D+1) were considered. The total number of spores and the number of infectious spores (i.e. germinated spores that have the ability to infect the plant) of each replicate were counted using a Malassez cell under microscope. For each replicate, five countings were used to take into account an effect of "petri dish" nested within "NaCl concentration" factor. The mycelium dry weight was estimated after a desiccation period of one night in an oven at 105°C (Kranner et al., 2002). Desiccation was performed until stable weights of the samples were reached. To increase the effectiveness and statistical power, this experiment was performed two times following the same design and methodology, allowing to take into account an "experiment" effect.
Data analysis: ANOVAs were performed using univariate (mycelium daily growth) or multivariate (mycelium dry weight, sporulation rate, germination rate, number of infectious spores) hierarchical models. Corrected means, which take into account the effect of "petri dish" and "experiment" effect, were calculated using "LSmeans" R package while significant differences were assessed using Tukey LSD test. Correlation between various parameters and NaCl concentration were also studied using the Pearson correlation test for α=5 %. R version R-3.4.0 was used to perform all the ANOVAs, Tukey's and correlation tests.

RESULTS
Effect of NaCl concentration on the growth parameters of Foe: A significant NaCl effect was observed on the average radial daily growth (P<0.001). The average radial daily growth was significantly lower with the NaCl concentration 10 g/L than that of the three other concentrations. In addition, no significant differences were observed between the average radial daily growth under 0 g/L, 2.5 g/L, and 5 g/L (Table 1, Figure S1). The average radial daily growth under 2.5 g/L was 0.48 mm (6.2%) higher than that of concentration 10 g/L. A decrease in radial daily growth over time was observed with the various concentrations of NaCl. In addition, the highest differences occurred at the beginning of the incubation period, from D2 to D4 (Figure 1). Significant NaCl concentration effect was observed only on D2, D3, D4 (P< 0.001), D6 (P<0.05) and D9 (P<0.01) (Table 1, Figure 1). At D2, the radial daily growth was significantly higher with NaCl 0 g/L than with 2.5 g/Land 5 g/L, which was in turn significantly higher than that of 10 g/L. The radial daily growth for 0 g/L was 11.2 mm (122%) higher than for 10 g/L (Table 1, Figure S2). At D3, the radial daily growth was significantly higher with NaCl 2.5 g/L than with the three other concentrations, these later being not significantly different. The radial daily growth with 2.5 g/L was 5.9 mm (69%) higher than that under 5 g/L (Table 1, Figure S2). At D4, the radial daily growth with NaCl concentration of 0 g/L was significantly lower than with the three other concentrations, these later being not significantly different. The radial daily growth with 2.5 g/L was 5.7 mm (76%) higher than that of 0 g/L (Table 1, Figure S2). At D6, the radial daily growth with 10 g/L was significantly higher than that of 0 g/L, 2.5 g/Land 5 g/L while no significant difference was observed from 10 g/L to 2.5 g/L. The radial daily growth with 10 g/L was 4 mm (88%) higher than that of 2.5 g/L (Table 1, Figure S2). At D9, the radial daily growth with 5 g/Lwas significantly lower than that of 0 g/L and 2.5 g/L, these later being not significantly different. Radial daily growth with 10 g/L was not significantly different from that of the three other concentrations. The radial daily growth with 5 g/L was 3.3 mm (114%) higher than that of 2.5 g/L (Table 1, Figure S2). "Experiment" effect was significant for α=5% (P=0.003).
In addition, the mycelium dry weight were significantly higher with 0 g/L and 10 g/L than with 2.5 g/L and 5 g/L (P<0.001) (Table 1), with no significant differences within each of these two groups (Table 1, Figure S3).

Effect of NaCl concentration on the reproduction and infectiveness parameters of Foe:
The effect of NaCl on the sporulation rate with the concentrations 0 g/L and 2.5 g/L were significantly higher than that of 5 g/L, which was in turn significantly higher than that of 10 g/L (P<0.001). The sporulation rate for concentration 0 g/L was 220 % higher than that of 10 g/L (Table 2, Figure S4).
Germination rate under concentration 0 g/L and 2.5 g/L was significantly lower than that with 5 g/L and 10 g/L (P<0.001), with no significant differences within each of these two groups. Germination rate for concentration 5 g/L was 4.03% higher than that of concentration 2.5 g/L (Table 2, Figure S4). With regard to the infectious spores, the number of infectious spores was significantly higher with concentration 0 g/L and 2.5 g/L than that of 5 g/L and 10 g/L (P<0.001), with no significant difference within these two groups. A number of infectious spores for concentration 0 g/L was 112 % higher than that of 10 g/L (Table 2, Figure S4).  .14) Significance levels of P-values: *, 5 %; **, 1 % and ***, 1%. For each parameter, corrected means for the four concentrations are given. Corrected means with different letters are significantly different for α = 5 %.

DISCUSSION
This study evaluated the effects of four various concentrations of NaCl 0, 2.5, 5 and 10 g/L on the growth parameters of Foe through radial daily growth of mycelium and mycelium dry weight; the reproductive ability using sporulation rate and their infectiveness by assessing germination rate and infectious spores.

Effect of NaCl on growth parameters:
The effect of NaCl on the average radial daily growth was significantly lower with 10 g/L (P<0.001) than with the three other concentrations, which were not significantly different between themselves (Table 1, Figure S1). Average radial daily growth decreased with an increase in NaCl concentration, which was confirmed by the significant negative correlation observed (P<0.001; R 2 =-0.686) (Figure 2a). These results are consistent with those of Davet (1967), who found that low NaCl concentrations did not affect the mycelium growth of Fusarium oxysporum species. However, they are not aligned with the results of other authors. In fact, Daami-Remadi et al. (2009) observed no significant effect of NaCl on mycelium growth, whatever the concentration was. In addition, Besri (1981); Keren (2000); Regragui (2005) Above this threshold, a negative correlation between mycelium growth and NaCl concentration was observed. With regard to the NaCl effect on radial daily growth along the growth kinetics, more contrasting results were observed than with average radial daily growth. In fact, the significant effect of NaCl concentration was observed with radial daily growth, but not every day on the radial daily growth kinetics. Significant differences was noticed on D2, D3, D4 (P<0.001), D6 (P<0.01) and D9 (P<0.01) (Table 1, Figure 1, Figure S2). Even if significant effects were observed at different days along the kinetics (from D2 to D9), we clearly observed that the highest effects were concentrated at the beginning of the radial daily growth kinetics (D2, D3 and D4). This may be explained by the fact that microorganism can use several strategies to encounter osmotic stress, such as ionic pumps. Few pumps may be produced at the beginning of the kinetics and would not allow Foe to reject NaCl through its membrane and wall, leading to higher NaCl effect during the first incubation days. Also, it was observed at D2, D3 and D4 that an increase in NaCl led to a decrease in radial daily growth (Table 1, Figure1, Figure S2). However, the radial daily growth were significantly lower with 0 g/L than with the 2.5 g/L at D3 and D4. Thus, even if high NaCl concentrations led to lower radial daily growth, it seems that the presence of of a minimum amount of NaCl in the medium is beneficial to the fungus. This observation was also made by Regragui (2005) with Verticillium alboatrum, and may be due to the fact that the fungus may need a minimum amount of salt to develop and ensure physiological processes. However, along with the radial daily growth kinetics, we also observed contrasting results. In fact, at D6, radial daily growth increased with increasing NaCl concentrations, with a significant difference between 2.5 g/L and 10 g/L. This growth pattern was observed again at D9 with a significant increase in radial daily growth between 2.5 g/L and 5 g/L. This contrasting NaCl effect along the incubation period was not described by others authors but could be considered as a real biological/physiological process.
A significant effect of NaCl concentration was observed on the mycelium dry weight (P<0.001) (Table 1, Figure S3). Mycelium dry weight were significantly higher for concentrations 0 and 10 g/L than for 2.5 and 5 g/L, with an increase of 0.1g and 0.06g, respectively. It came out that the more the NaCl concentration was, the less was the mycelium dry weight. This was confirmed by a significant negative correlation (P<0.01; R 2 =-0.418) (Figure 2b). This result is contradictory with that of Sanogo (2004) who observed an increase in mycelium dry weight with an increase in NaCl concentration in Phytophthora capsici. However, those findings was consistent with Daami-Remadi et al. (2009) who observed a decrease in mycelium density of Verticillium dahliae with an increase in NaCl concentration of irrigation water. In the same way, Regragui (2005) observed no effect on mycelium dry weight for NaCl concentrations comprising between 0 and 10 g/L, but a regular decrease above 10 g/L.

NaCl effect on reproduction and infectiveness traits:
Sporulation rates were significantly higher with NaCl concentrations 0 and 2.5 g/L than with 5 g/L, this later being significantly higher than that of 10 g/L (Table 2, Figure S4). Foe sporulation decreased with increasing NaCl concentration and this was confirmed by the significant negative correlation observed (P<0.001; R 2 = -0.713) (Figure 2c). This result was contradictory to other authors' observations. In fact, Regragui (2005); Daami-Remadi et al. (2009) and Boumaaza et al. (2015) observed stimulation of sporulation rate with Verticillium alboatrum, Verticillium dahliae and Botrytis cinerea when NaCl concentration increased. However, our result is consistent with those of Sanogo (2004) who observed a decrease in sporangium production Phytophthora capsici with an increase in NaCl concentrations. A significant NaCl effect was observed on the germination rate (P<0.001) ( Table 2). In fact, germination rates were significantly lower with NaCl concentrations 0 and 2.5 g/L than with 5 and 10 g/L, without significant differences within these two groups (Table 2, Figure S4). Germination rate increased with increasing NaCl concentrations, which was confirmed by the significant positive correlation observed (P<0.001; R 2 =0.361) (Figure 2d). This result is contradictory to those of Sanogo (2004); Boumaaza et al. (2015) and Ragazzi and Vecchio (1992) who reported respectively a negative effect of NaCl on germination rate of Phytophthora capsici, Botrytis cinerea and Fusarium oxysporum f. sp

vasinfectum.
The number of infectious spores was significantly higher (P<0.001) with NaCl concentration of 0 and 2.5 g/L than with that of 5 and 10 g/L, without significant differences within each group (Table 2, Figure S4). The number of infectious spores decrease with a decrease in NaCl concentration, which was confirmed by the significant negative correlation (P<0.001; R 2 =-0.547) (Figure 2e). Variation between growth parameters: A decrease in both radial growth and mycelium dry weight was observed with the increase in NaCl. This suggests that even if NaCl impacted negatively Foe growth, it would not impact mycelium density although this could be the case (Daami-Remadi et al., 2009). In fact, a decrease in radial growth without modification of mycelium dry weight would result in an increase in mycelium density (i.e. wall thickening). It was not the case with Foe since both parameters decrease with the increase in NaCl concentrations, suggesting a stable mycelium density. However, the stronger negative correlation between NaCl concentration and radial growth (P<0.001) than between NaCl and mycelium dry weight (P<0.01) might reflect that they did not strictly covariate, thus leading to an increase in mycelium density. In addition, there was no evidence that other morphological parameters (branching) were not affected by NaCl. These aspects would need deeper investigations (microscopic observations) in order to be able to study the effect of NaCl on Foe morphology. Variation between reproduction and infectiveness parameters: NaCl may act on reproduction and infectiveness parameters of a phytopathogenic fungus, with more or fewer constraints. Thus, NaCl effect on the number of infectious spores may vary depending on its effect on parameters that compose this composite trait that is sporulation rate and germination rate. Indeed, an increase of sporulation rate with the increase in NaCl concentration, at a constant germination rate, would lead to a higher number of infectious spores and controversely. In the case of an NaCl effect on one trait or the other, it may lead to a positive or negative impact in infectiveness, depending on the respective effects of NaCl on these two traits. In the case of Foe, we observed that NaCl led to a decrease in infectiveness. In fact, despite a positive NaCl effect on the germination rate, the decrease in sporulation rate is too high to be compensated. Variation between growth, reproduction and infectiveness parameters: NaCl impacted both growth, reproduction and infectiveness parameters. NaCl impacted negatively all the studied parameters, except the germination rate. Regragui (2005) also observed that NaCl had an effect on both reproduction, sporulation and infectiveness parameters with Verticillium albo-atrum, but that effect was positive. Other authors observed that NaCl impacted these parameters in various ways. In fact, Sanogo (2004) observed with Phytophtora capsici that NaCl stimulated growth but impacted negatively reproduction parameters while Daami-Remadi et al. (2009) showed that NaCl did not affect mycelium growth in Verticillium dahliae but reduced mycelium density and stimulated sporulation. Toward potential management of Foe in oil palm plantations using NaCl: Basically, our results showed that NaCl has a negative impact on growth, reproduction and infectiveness trait of Foe, and thus could be potentially used as a method of management of the disease in the field. However, two major points have to be taken into account and need further investigations. Firstly, we have to ensure that the negative effects observed in vitro on Foe traits lead to an effective decrease of the disease incidence on the host plant, by performing in vivo trials on plantlets in the nursery. Secondly, soil salinity is known to have a negative effect on plant growth. Indeed, suppression/ reduction of growth in salty soils occurs in all plants, but their tolerance levels and rates of growth reduction vary widely among plants (Alia et al., 1995;Yoshiba et al., 1997;Hayashi and Murata, 1998;Yeo, 1998;Sánchez et al., 1998;Ramoliya et al., 2004;Flowers, 2004;Sánchez-Blanco et al., 2004;Parida and Das, 2005;Benlloch-González et al., 2005;Ghadiri et al., 2006). Usually, this suppression/ reduction of growth is followed by limitation in plant productivity. This negative effect of soil salinity has been reported for oil palm by Henry and Wan (2012). They observed that fresh fruit bunch yields from fields with salty soils were significantly lower to normal areas, with 16.5 t ha -1 and 23.5 t ha -1 , respectively. In the same way, the oil-to-bunch was found to be significantly affected by salinity, ranging from 16.5%-18% in saline areas to 22.2%-22.4% in non-saline areas. This implies that even if a positive effect on Foe incidence was observed in vivo trials, it might be counterbalanced by a negative effect on oil palm growth and yield in the field. Thus, such trade-off would require field experiments to assess the best concentration to maximize the positive NaCl effect on disease incidence while minimizing the negative effect on productivity.

CONCLUSION
In this study, we assessed the effect of NaCl on growth, reproduction and infectiveness traits of the oil palm wilt fungus, Fusarium oxysporum f. sp. elaeidis. Also, we showed that NaCl had a negative effect on growth parameters, by reducing the radial daily growth and mycelium dry weight. There was, for instance, no evidence that NaCl also impact mycelium density, but this hypothesis would require further investigations. In addition, we observed a negative effect of NaCl on reproduction and infectiveness parameters: a reduction in sporulation rate, an increase in germination rate while resulting in a reduced number of infectious spores. Thus, NaCl basically impacts in a negative way the development and the fitness of this pathogenic fungus. The present work gives rise to some perspectives in terms of management of Foe in the field, and open ways to future work to assess the sustainability of such control method in oil palm plantations. NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (g.L -1 ) NaCl concentration (