Interacting Abiotic Factors Affect Growth and Mycotoxin Production Profiles of Alternaria Section Alternaria Strains on Chickpea-Based Media

Chickpea is susceptible to fungal infection and mycotoxin contamination. Argentina exports most of its chickpea production; thus, its quality is of concern. The Alternaria fungal genus was found to be prevalent in chickpea samples from Argentina. The species within this genus are able to produce mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). In this context, we evaluated the effect of water activity (0.99, 0.98, 0.96, 0.95, 0.94, 0.92, and 0.90 aW), temperature (4, 15, 25, and 30 °C), incubation time (7, 14, 21, and 28 days), and their interactions on mycelial growth and AOH, AME, and TA production on chickpea-based medium by two A. alternata strains and one A. arborescens strain isolated from chickpea in Argentina. Maximum growth rates were obtained at the highest aW (0.99) and 25 °C, with growth decreasing as the aW of the medium and the temperature were reduced. A. arborescens grew significantly faster than A. alternata. Mycotoxin production was affected by both variables (aW and temperature), and the pattern obtained was dependent on the strains/species evaluated. In general, both A. alternata strains produced maximum amounts of AOH and AME at 30 °C and 0.99–0.98 aW, while for TA production, both strains behaved completely differently (maximum levels at 25 °C and 0.96 aW for one strain and 30 °C and 0.98 aW for the other). A. arborescens produced maximum amounts of the three toxins at 25 °C and 0.98 aW. Temperature and aW conditions for mycotoxin production were slightly narrower than those for growth. Temperature and aW conditions assayed are those found during chickpea grain development in the field, and also could be present during storage. This study provides useful data on the conditions representing a risk for contamination of chickpea by Alternaria toxins.


Introduction
Since ancient times, chickpea (Cicer arietinum L.) has been an important crop, particularly in Asia, although over the years, it has been adapted to many continents including the Americas. The genus Cicer, which includes the cultivated chickpea, belongs to the family Fabaceae, subfamily Papilionoideae. Like other pulses, chickpeas are well known for their soil fertility restoration value due to their ability to fix nitrogen, helping agricultural systems to be productive and sustainable. Furthermore, chickpeas are highly nutritious, having high protein, fiber, and fat contents, but a lower carbohydrate content than wheat. Chickpea plants also contain many bioactive compounds, such as phenolic acid and isoflavones [1].
Globally, soybeans lead legume production, followed by peanuts, dried beans, dry peas, and chickpeas, among others [2]. In recent years, the global demand for legumes has increased, and this trend is expected to continue in the future. The average consumption of legumes in the world is about 8 kg per capita per year [3], while that of Argentina reaches barely 800 g per capita per year [4]. Most of the Argentinean chickpea production The objective of the present study was to determine the effects of a W and temperature on the growth and production of AOH, AME, and TA by A. alternata and A. arborescens on a synthetic chickpea media. As a result, we provide more information about the influence of environmental factors on the contamination of this pulse with Alternaria toxins

Fungal Strains
Three Alternaria section Alternaria strains (A. alternata RC-CR 767 and RC-CR 902; A. arborecens RC-CR 910), previously isolated from chickpeas in Argentina during the 2018 harvest season, were used. They were previously identified by morphological and molecular criteria [30][31][32][33][34]. For the molecular characterization, the 1-α elongation factor (tef1), Alternaria major allergen (Alt-a1), and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were partially sequenced indicating that these isolates belong to the section Alternaria and can be characterized as A. alternata and A. arborescens. The ability of the strains to produce AOH, AME, and TA was determined in ground rice-corn steep liquor medium and detected by high-performance liquid chromatography (HPLC) [10]. All the strains are preserved at the Universidad Nacional de Río Cuarto (UNRC) culture (RC) collection as spore suspensions in 15% glycerol frozen at −80 • C.

Media Preparation
Chickpeas harvested in Argentina were finely milled by using a Romer mill (Romer Labs Inc., Union, MO, USA). Mixtures of 2% (w/v) of milled chickpea in water were prepared, and 2% (w/v) agar (technical agar no. 2, Oxoid, Basingstoke, UK) was added. The a W of the media was modified by adding mixtures of water/glycerol instead of water to obtain target a W treatments of 0.99, 0.98, 0.96, 0.95, 0.94, 0.92, and 0.90 before autoclaving for 15 min at 121 • C [35]. The molten cooled media were vigorously mixed before pouring into 9 cm Petri plates (20 mL per plate), cooled, and stored at 4 • C in sealed bags prior to use. The a W of representative samples of media was checked with an Aqualab Series 3 (Decagon devices, Inc., Pullman, WA, USA). In order to detect any significant deviation of the media a W values, uninoculated control plates were prepared and measured at the end of the experiment.

Inoculation, Incubation, and Growth Assessment
Chickpea-based media were centrally inoculated with a mycelial plug of 3 mm diameter and removed with a sterile punch from the active growth area of each Alternaria section Alternaria strain grown on the synthetic nutrient agar (SNA) at 25 • C for seven days. After inoculation, plates with an equal a W were placed in polyethylene bags and incubated at 4, 15, 25, and 30 • C for a period of 28 days.
A full factorial design with 3 replicates was applied. The factors analyzed were strains (3) × a W levels (7) × temperature (4), and as a response, the growth rate was analyzed.

Mycotoxin Analysis
For mycotoxin extraction after 7, 14, 21, and 28 incubation days, three agar plugs (4 mm diameter) were cut with a hole punch from the edge of a colony from each Petri plate, weighed, and placed in a 4 mL amber vial. The extraction method used for the three toxins was the microscale extraction technique proposed by Smedsgaard [36] modified in three extraction steps suitable for the mycotoxin produced by species of the genus Alternaria [37].
AOH, AME, and TA were detected and quantified by HPLC using the equipment and settings described by Nichea et al. [10]. The recovery rate of the method was >80% when different amounts of Alternaria mycotoxins (AOH, AME, and TA) were added (range 0.1 to 10 µg/g). For the three toxins, the limit of detection (LOD; signal-to-noise ratio 3) was 0.01 µg/g, and the limit of quantification (LOQ) was established as three times the detection limit.

Statistical Analysis
The growth rates and mycotoxin concentrations (transformed to Log 10 ) were evaluated by analysis of variance (ANOVA) using SigmaStat for Windows version 2.03 (SPSS Inc., Chicago, IL, USA). Statistical significance was judged at the level p ≤ 0.001. When the analysis was statistically significant, the post hoc Tukey's multiple comparison procedure was used for separation of the means (LSD-Fisher p < 0.05). Surface response and contour map graphs were performed using Sigma Plot v. 10.0 (Systat Software Inc., Hounslow, London, UK).

Effect of a W and Temperature on Growth Rates
The results obtained for the growth rates of the three strains studied, under different a W and temperature conditions, are presented in Figure 1. Both factors, a W and temperature, influenced the mycelial growth rate of the three Alternaria section Alternaria species under study. The data show that A. arborecens RC-CR910 presented the highest growth rate followed by A. alternata RC-CR902 and finally by A. alternata RC-CR767. In general, the response of the growth rate to a W and temperature was similar for both species. Maximum growth rates were observed at the highest a W (0.99) evaluated at 25 • C, and they declined when the water availability in the media decreased. Regarding incubation temperature, higher growth rates were observed at 25 • C, decreasing in the following order: 30, 15, and 4 • C. Moreover, all of the strains under study were unable to grow at a W levels ≤ 0.92 at 4 • C.
An analysis of variance of the effect of a single factor (a W , temperature, and strain) and its double and triple interactions revealed that all factors and their interactions had a significant influence on the growth rate. The factor that influenced the fungal growth the most was temperature followed by a W and the temperature × a W interaction (Table 1). A posteriori analysis showed that the strains, a W levels, and the different temperatures studied were significantly different from each other (LSD-Fisher p < 0.05).
With the growth rate data obtained from the three strains evaluated (two A. alternata and one A. arborescens) under different growing conditions, contour maps were generated in an attempt to identify the optimum and marginal a W and temperature conditions for growth ( Figure 2). As both A. alternata behaved similarly, just one contour map is shown for this species. From these contour maps, the optimum conditions, regardless of the species, is around 20-30 • C at 0.99-0.95 a W . On the other hand, marginal conditions for growth are over a wider temperature range and limited to about 0.92-0.90 a W levels.

Effects of a W , Temperature, and Incubation Time on Mycotoxin Production
Figures 3-5 show the effects of a W , temperature, and incubation time on AOH, AME, and TA production by both Alternaria species grown in a chickpea-based media over 21 days. The results of this study show that the time course of production of the toxins evaluated varied with temperature and a W . Statistically, the effects of a W , temperature, and incubation time and their interactions were found to be significant ( Table 2).
In general, the production of the three toxins increased with incubation time. The maximum production was reached at 14/21/28 days, depending on the toxin. Overall, the toxin concentrations produced by A. alternata RC-CR902 were higher than those produced by A. alternata RC-CR767 and A. arborescens RC-CR910 under almost all the tested conditions.
We will present the results separately for both Alternaria species, starting with both A. alternata strains.
AOH maximum production for A. alternata RC-RC902 was reached with temperatures of 25 and 30 • C at 0.98 a W after 14 and 28 days of incubation ( Figure 3). On the other hand, for the strain RC-CR767, the maximum level was obtained at 30 • C after 14 days of incubation at the highest a W tested (0.99), followed by 25 • C at 0.96 a W after 28 days of incubation. At 15 • C, a significant reduction in AOH production was observed for both strains. At the lowest temperature tested (4 • C), the production of this mycotoxin was almost inhibited; it was only detected, at very low levels, with 0.99 and 0.98 a W , after 28 days of incubation.

Effects of aW, Temperature, and Incubation Time on Mycotoxin Production
Figures 3-5 show the effects of aW, temperature, and incubation time on AOH, AME, and TA production by both Alternaria species grown in a chickpea-based media over 21 days. The results of this study show that the time course of production of the toxins evaluated varied with temperature and aW. Statistically, the effects of aW, temperature, and incubation time and their interactions were found to be significant ( Table 2).
In general, the production of the three toxins increased with incubation time. The maximum production was reached at 14/21/28 days, depending on the toxin. Overall, the toxin concentrations produced by A. alternata RC-CR902 were higher than those produced by A. alternata RC-CR767 and A. arborescens RC-CR910 under almost all the tested conditions.
We will present the results separately for both Alternaria species, starting with both A. alternata strains.
AOH maximum production for A. alternata RC-RC902 was reached with temperatures of 25 and 30 °C at 0.98 aW after 14 and 28 days of incubation (Figure 3). On the other Figure 2. Two-dimensional contour maps of Alternaria alternata RC-CR767 and Alternaria arborecens RC-CR910 growth profiles in relation to temperature and water activity. Similar colors refer to similar growth rates (mm/day).
The pattern of AME production was similar to those observed for AOH production by the two A. alternata strains (Figure 4). The highest AME production by the strain RC-CR767 was obtained at 0.99 a W and 30 • C after 14 days of incubation, in comparison to the strain RC-CR902, which presented the highest level at 0.98 a W and 30 • C after 28 days of incubation. At temperatures of 30 • C and 25 • C, the strain RC-CR902 showed high production levels at 0.98 a W , and mycotoxin concentration increased during the incubation time, while the strain RC-CR767 presented the most favorable conditions at 0.99 a W at 30 • C and 0.96 a W at 25 • C. At 15 • C, a significant reduction in AME production occurred for the two strains under study. At 4 • C, AME production was detected at trace levels of 0.99 and 0.98 a W .
For TA production, a maximum concentration was obtained for A. alternata RC-CR 797 at 0.96 and 0.94 a W and 25 • C after 21 and 14 days of incubation, respectively ( Figure 5). RC-CR902 produced the maximum TA level at 0.98 a W after 14 days of incubation at the highest temperature tested. In addition, for this strain at 15 • C, high levels of TA production were observed at 0.99, 0.98, and 0.96 a W in comparison with the other A. alternata strain. At the lowest temperature tested (4 • C), a decrease in production levels was observed. However, these were still high compared to those observed for AOH and AME, especially by the strain RC-CR902 at ≥0.94 a W .              In relation to A. arborescens, this strain reached the maximum production of AOH, AME, and TA at 0.98 a W and 25 • C after 28 days of incubation. Considering the incubation temperature, the production of the three toxins was higher at 25 • C, decreasing in the following order: 30, 15, and 4 • C. At the lowest temperature tested (4 • C), no AME production was observed regardless of the a W , but traces of AOH and TA were detected at 0.99 and 0.98 a W after 28 days of incubation.
The mycotoxin (AOH, AME, and TA) production data obtained were used to develop contour maps in order to identify the optimal and marginal conditions of a W and temperature and the range of conditions for mycotoxin production after 28 days of incubation ( Figure 6). Figure 6. Two-dimensional contour maps of alternariol, altenariol monomethyl ether, and tenuazonic acid production by Alternaria section Alternaria strains: Alternaria alternata RC-CR767, Alternaria alternata RC-CR902, and Alternaria arborecens RC-CR910 in relation to temperature and water activity after 28 days of incubation. The numbers on the scale refer to toxin levels (µg/g).

Discussion
To our knowledge, this study represents the first approach to understanding the interaction effect of aW and temperature conditions on the growth and mycotoxin production by two Alternaria species belonging to section Alternata (A. alternata and A. arborescens) on chickpea-based media. It is important to remark that the strains used in the present Figure 6. Two-dimensional contour maps of alternariol, altenariol monomethyl ether, and tenuazonic acid production by Alternaria section Alternaria strains: Alternaria alternata RC-CR767, Alternaria alternata RC-CR902, and Alternaria arborecens RC-CR910 in relation to temperature and water activity after 28 days of incubation. The numbers on the scale refer to toxin levels (µg/g).

Discussion
To our knowledge, this study represents the first approach to understanding the interaction effect of a W and temperature conditions on the growth and mycotoxin production by two Alternaria species belonging to section Alternata (A. alternata and A. arborescens) on chickpea-based media. It is important to remark that the strains used in the present study were previously isolated from chickpea produced in Argentina and identified using multigenic phylogenetic analyses [10]. All these details make the results obtained more reliable, because the taxonomy of the Alternaria genus is quite controversial. Both species A. alternata and A. arborescens share morphological features and are very difficult to be differentiated using traditional methods. This could be the reason why all the strains were classified in the past as A. alternata, thus leading to a general belief that it is the most common species isolated in food products.
Chickpea is sown in Argentina between April and June during the autumn season; thus, part of the crop cycle coincides with a gradual decrease in average temperature and luminous intensity, a shortened photoperiod, and scarce rainfall. The rest of the cycle (mainly the reproductive phase) unfolds under a continuous increase in temperature, luminous intensity, and rainfall [38]. The optimum temperature for the chickpea's reproductive phase ranges from 5 • C to 30 • C. For all these reasons, the temperatures used in the present study where chosen to simulate the conditions in which this crop grows in the fields. In addition, the a W values used in the present study cover the fluctuation of this parameter during early seed formation (R5), until the seeds reach full physiological maturity (R8) [39]. On the other hand, these conditions of a W and temperatures can also be present during inappropriate storage.
In the present study, not only temperature, but also a W affected growth, and the pattern obtained was independent of the strains/species evaluated. Optimal a W levels for growth were 0.99 to 0.95, with an optimal temperature ranging between 20 and 30 • C and the highest growth rates for the three strains at 0.99 a W and 25 • C. We defined as optimal those growth rates that were above 50% of the maximum growth rates obtained. No growth was observed at ≤0.92 a W at 4 • C.
Previous studies about the requirements of water and temperature for A. alternata growth have revealed that this fungus requires a minimum of 0.84-0.88 a W , and optimal growth has been observe at 0.98 to 1.0 a W at 25 • C [26][27][28][29]40,41].
The effect of environmental conditions on A. arborescens growth has not been previously studied in depth. Only few attempts can be found in the literature. Vaquera et al. [40] studied the effect of a W and temperature on the growth of one strain of A. arborescens in a tomato-based media. They informed that the highest growth rate was observed at 0.995 a W and 30 • C (7.21 mm/day), while the lowest occurred at 0.950 a W and 6 • C, but they did not find statistical differences between the growth rates obtained at 0.975 a W at 30 • C and 0.995 a W at 25 and 30 • C. Camiletti et al. [42] studied the effect of temperature (10,15,20,25,30, and 35 • C) on mycelial growth of A. arborescens and A. alternarta strains associated with preharvest fruit drop in California citrus in Potato Dextrose Agar (with freely available water ≈ 0.995 a W ). They observed that A. alternata had the capacity to grow faster than A. arborescens at all the evaluated temperatures, except at 25 and 35 • C, when the growth patterns were similar for both species. Our results for A. arboresces are in agreement with those obtained by Oviedo et al. [41] and partially agree with those informed by Camiletti et al. [42]. It is important to point out that, according to our results, A. arboresces grows significantly (p < 0.05) faster than both A. alternata strains, even though the growth patterns obtained through all the conditions were the same.
In the present study, both variables (a W and temperature) affected mycotoxin production, and the pattern obtained was dependent on the strains/species evaluated. In general, both A. alternata strains produced maximum amounts of AOH and AME at the highest temperature analyzed, 30 • C and 0.99-0.98 a W . For TA production, both A. alternata strains behaved completely different; RC-CR76 maximum levels were obtained at 25 • C and 0.96 a W , and for RC-CR902, the conditions were 30 • C and 0.98 a W . For A. arborescens, maximum AOH, AME, and TA production occurred at the same conditions: 25 • C and 0.98 a W . In general, all these a W and temperature conditions were not optimal for growth and were slightly narrower.
There are several studies in the literature dealing with the effect of a W and temperature on mycotoxin production by A. alternata strains on natural substrates or on media-based natural substrates [26][27][28][29]41,43], but only a few have been performed with A. arborescens [40,44]. It is important to remark that none of them have been performed on chickpea. However, in general, the results of the present work are similar to those obtained by the authors mentioned above.
Although the basic culture media (chickpea, soybean, grape, tomato pulp, etc.) may vary, mycotoxin production tends to be similar under controlled environmental conditions. Because Alternaria section Alternata species (mainly A. alternata) are isolated with a high frequency in chickpea in Argentina and worldwide [8] and the natural occurrence of AOH and AME in this substrate has been informed, the contour maps developed with the results of the present study can provide very useful information to effectively facilitate risk prediction about growth and mycotoxin production during chickpea grain development, harvesting, and storage. All the reported information is valid because the a W and temperature ranges used in these assays simulated the ranges that occur during chickpea development in the field and those that can be found during improper storage.

Conclusions
So far, there is insufficient monitoring data on Alternaria mycotoxin occurrence and an absence of statutory or guideline limits set for these compounds in foods and feeds by regulatory authorities worldwide. However, there is increasing awareness among European authorities and the scientific community about the high incidence rate of Alternaria toxins in relevant foodstuffs and about population exposure through diet [45]. It is well known that the presence of Alternaria toxins in food can be reduced or prevented by using good agricultural practices, good storage and transport conditions, and good manufacturing practices. The present study provides useful information in that direction.