Evaluation of some techniques for drying conidiated rice culture of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. with a bioassay evaluating their eligibility

Abstract

Background

Production of entomopathogenic fungi on a solid substrate, mostly on rice, is preferred by many producers due to its low technological requirements. Drying may be needed after fermentation to preserve the viability of the conidia until use. This study aimed to introduce some uncomplicated drying techniques and study their effectiveness in terms of drying time and extent, as well as their impact on the quantity of conidia produced and their biological activity. Four techniques were studied, i.e., condensing water vapor (by cooling), desiccant (using NaOH), air drying on a net, and air drying on a solid (impermeable) surface.

Results

Condensing water vapor and desiccant techniques were the fastest and most efficient, resulting in the lowest moisture content, ranging between 12 and 13%. While drying on a solid surface was the slowest and resulted in the highest moisture content of 32.98%. All of the dried samples yielded a lower quantity of conidia compared to the fresh samples. Samples of condensing water vapor, desiccant, and net techniques yielded approximately a similar quantity of conidia, ranging between 74 and 71% of the yield of the fresh sample. The solid surface sample yielded the lowest quantity of conidia, representing 63.78% of the fresh sample yield. A bioassay test on the mite, Tetranychus urticae Koch (Acari: Tetranychidae), showed that conidia dried via condensing water vapor and desiccant techniques kept their pathogenicity similar to the fresh sample, while samples dried on a net and solid surface significantly lost part of their pathogenicity.

Conclusion

The condensing water vapor and desiccant techniques were the most efficient regarding the time needed, conidia yield, and pathogenicity. Drying on a solid surface was the least effective regarding the same factors.

Background

Usually, farmers face great challenges due to damage caused by insects and mites. Throughout the course of history, farmers have dedicated considerable efforts to control or manage these pests (Oerke 2006). Pesticides may appear as a convenient and swift solution. However, the harms caused by pesticides are no longer hidden nor a matter of discussion (Tudi et al. 2021). Biological control strategies may offer a suitable alternative; however, challenges persist in terms of producing and applying biocontrol agents. Consequently, it is imperative for scientific research efforts to be directed toward overcoming these challenges (Daniels et al. 2023). Within this context, this study aimed to achieve progress in a specific facet of these efforts.

Entomopathogenic fungi play a remarkable role as biocontrol agents, being produced by both large and small scale producers. Fermentation on a solid substrate (commonly rice) is suitable for low-technology artisanal producers of entomopathogenic fungi (Mascarin and Jaronski 2016). After the fermentation process, the spores can be washed away from the rice media, forming an aqueous suspension for application. It is essential to utilize this suspension immediately, as any postponement would lead to loss of the spores by germination (Sala et al. 2019). When there is no immediate requirement for spore utilization, it is necessary to dry sporulated cultures to facilitate the separation of spores from the substrate and maintain their viability, pathogenicity, and ability to germinate for a long time (Latifian et al. 2013). Commonly, air drying is used; however, it is time-consuming process that can extend up to 10–12 days. This extended duration may result in vegetative regrowth and a subsequent loss in spore yield (Mathulwe et al. 2022).

The objective of this study was to simplify the production of entomopathogenic fungi by proposing and introducing simple and practical drying techniques and then investigating and comparing these techniques with the conventional air drying technique. The purpose of this research is to accelerate and improve the efficiency of the drying process, as well as decrease conidia loss during the drying process via uncomplicated techniques.

Methods

Conidia production

An isolate of the entomopathogenic fungus, Beauveria bassiana (Bals.) Vuill., previously examined by Ali et al. (2020), was acquired from the author and cultivated using the following procedure. Erlenmeyer flasks (1000 ml) containing 100 g of previously prepared boiled rice were sterilized in an autoclave for a duration of 20 min at a temperature of 121 °C. After cooling to room temperature, the flasks were inoculated with 1 ml of conidia suspension, containing 1 × 10⁸ conidia/ml. The inoculated flasks were then incubated for 2–3 weeks in darkness at a temperature of 25 ± 1 °C until the development of conidiated cultures.

Preparation of samples and drying techniques

After the incubation period, a sufficient number of flasks were opened to obtain the fermented rice. The contents of these opened flasks were mixed and then distributed into 24 divisions of 100 g each. Sixteen of these samples were taken to be subjected to the four drying techniques, four replicates for each. Additionally, four samples were evaluated in their fresh state, while another four samples were subjected to oven drying for a duration of 24 h at a temperature of 105 °C to obtain the dry weight of the fermented rice. Accordingly, the moisture content of the other dried samples could be calculated subsequent to the completion of each drying technique.

Condensing water vapor technique

This technique depended on reducing the humidity in the ambient air by condensing the water vapor. It was carried out in a traditional incubator consisting of a cabin with a size of 216 L, equipped with a refrigeration system, heater, and tangential fan, all of which were controlled by a thermostat. The incubator was modified so that the heater and the fan were connected directly to the electric source bypassing the thermostat, keeping only the refrigeration system under the control of the thermostat. The heater was also replaced by a smaller 60 W one, which was found to be adequate as a higher heater power, when it works continuously, may generate more heat than the capability of the refrigeration system to control the temperature. Keeping the heater running continuously raised the temperature, subsequently driving the cooling system to run more frequently and for longer durations, resulting in the trapping and condensation of water vapor on the freezer body. A funnel-shaped shelf was placed under the body of the freezer to collect the condensed water droplets and direct them to a bottle with a narrow opening to prevent re-evaporation of the water. These modifications allowed the achievement of low relative humidity levels at relatively low temperatures. A relative humidity level of 18–22% was achieved by setting the thermostat to 30 °C. Four samples (100 g each) were distributed on (20 × 30 cm) plastic nets positioned on incubator’s racks and left to dry. A schematic diagram of the incubator is presented in Fig. 1.

Fig. 1

Schematic diagram of the used incubator

Desiccant technique

Four airtight plastic boxes with a base measuring (50 × 30 cm and 30 cm) in height were used. An amount of one hundred grams of sodium hydroxide, serving as a desiccant, was deposited in a plastic tray placed at the bottom of each box. Samples were dispersed on net shelves (20 × 30 cm), fixed in the middle of each box (15 cm) above the sodium hydroxide tray. A small electric fan (12 cm) was affixed to one of the inner sides of each box to stir the air within. The boxes were closed and left at the room temperature of 27 ± 5 °C. Due to the presence of Sodium hydroxide in this system, a relative humidity level of 20–23% was achieved inside the enclosures.

Air drying on a netted surface

Four samples, weighing 100 g each, were dispersed onto plastic nets measuring (20 × 30 cm), mounted on (20 cm) high stands, and left exposed to the ambient air at room temperature of 27 ± 5 °C and 40–50% relative humidity.

Air drying on a solid (impermeable) surface

The samples (100 g each) were dispersed on a plastic trays measuring (20 × 30 cm) and exposed to the ambient air under the same conditions mentioned in the previous paragraph. All the aforementioned samples were weighed at 6, 12, 24, 48, 72, 96, 120, and 144 h intervals. Subsequently, the samples were collected and grounded carefully and intermittently to avoid any temperature rise caused by grinding. The resulting powders were then stored in airtight jars until the bioassay test.

Preparation of conidia’ suspension

After the drying process, the drying ratio was calculated for each of the above techniques, based on the calculated dry weight. Accordingly, the weight equivalent to 10 g of the original fresh sample was assessed for all the dried samples. The equivalent weights were 1.75, 1.76, 1.90, and 2.29 g for condensing water vapor, desiccant, air drying on a net, and air drying on a solid surface, respectively. In other words, the ten-gram equivalent weight was the remaining weight after subjecting a ten-gram fresh sample to the drying technique.

The amount of 10 g fresh fungus culture or the equivalent weight of each dried sample was suspended in 25 ml distilled water with 0.1% Tween 80. Taking into account adding an extra amount of distilled water to the dried samples equal to the amount of water lost during the drying process. By doing so, a comparable similar volume of suspension could be obtained from both the fresh and dried samples. Then, suspensions were filtered through sterile cheesecloth. The conidia were counted in the suspensions using a hemocytometer (Neubauer improved HBG, Germany 0.100 mm² × 0.0025 mm²) under a microscope to get the conidia’ number per gram fresh sample or its equivalent weight of dried samples.

Rearing of Tetranychus urticae Koch (Acari: Tetranychidae)

A susceptible colony of the red spider mites T. urticae was obtained from the acarology laboratory in Plant Protection Research Institute, A.R.C, Egypt. Mites were reared and maintained on detached Acalypha marginata leaves placed on moist cotton in arenas (20 × 12 cm) at 25 ± 1 °C. The cotton pads were moistened daily to avoid leaves dryness and to prevent mite escape. Leaves were changed by fresh ones from time to time when it is needed.

Bioassay test

The biological activity of the conidia resulting from the aforementioned drying techniques was evaluated against the spider mite, Tetranychus urticae Koch in order to be compared with the fresh conidia. A concentration of 2.86 × 10⁷ conidia/ml was prepared from each sample. This concentration was previously stated as the LC₅₀ value of the same fungus strain against the same strain of the mite T. urticae by Ali et al. (2020). This concentration was used as a standard to compare the biological activity of the resulting conidia. Ten newly emerged adult females of T. urticae mite were transferred to the surface of A. marginata leaf disks (2.5 cm diameter). Ten disks were used as replicates for each treatment. The disk surfaces carrying the mites were sprayed with the concentration where the spray was distributed homogeneously using a glass atomizer at the rate of deposit 2 ml/400 cm². Control disks were treated with 0.1% Tween 80 in distilled water. Then, disks were placed on moist cotton wool in Petri dishes and kept in an incubator at a constant temperature of 25 ± 0.5 °C and 60% ± 5 R.H for six days. Then, the mortality was counted and corrected according to the control by Abbott (1925).

Data analysis

Analysis of variance (ANOVA), single factor, was applied via Microsoft Excel 2010, and LSD was calculated to determine the significant differences among the different treatments.

Results

Drying rate

The results in Figs. 2 and 3 showed that drying techniques varied in terms of both the rate and extent of drying. Condensing water vapor and desiccant techniques exhibited a higher rate of drying than other techniques, as the samples subjected to these techniques lost more than 80% of their weight within 24 h. Furthermore, samples subjected to both of these techniques reached a final weight of approximately 17.5% of the original weight, with a moisture content of around 12–13% at the end of the experiment. The sample dried on the net needed three days to lose 80% of its weight and finally reached 18.97% of its initial weight with a moisture content of 19.14%, while the sample dried on a solid surface exhibited the slowest drying rate, as it failed to lose 80% of its weight, even after six days. Eventually, it reached 22.89% of its original weight with 32.98% moisture content. Data analysis of the final results and the LSD value of 2.97 indicated non-significant differences among the final weights of the first three methods, while drying on a solid surface was significantly less efficient. The moisture content of the above samples was calculated based on the oven sample, which showed that the dry weight of the studied samples was 15.34% of the original conidiated culture weight.

Fig. 2

Drying rate over time for the studied techniques

Fig. 3

Final weight and moisture content after subjecting 100 g fresh samples to the drying techniques

Conidia count

Obtained conidia were counted in order to assess any decrease in the amount of conidia retrieved after the drying process. Figure 4 illustrates the conidia count per gram of fresh conidiated rice along with the conidia count per gram equivalent weight from each dried sample. The fresh sample yielded a conidia count of 4.89 × 10⁷ per gram. According to the calculated LSD value of 1.14 × 10⁶, it was noted that all the dried samples returned significantly less amount of conidia than the fresh sample. Moreover, non-significant variations were detected among the samples resulting from the three drying methods, i.e., condensing vapor, desiccant, and air drying on a net, whereas these samples returned 3.45 × 10⁷, 3.57 × 10⁷, and 3.61 × 10⁷ conidia per gram equivalent, respectively. Samples subjected to these techniques lost about 26–29% of conidia than the fresh sample. The sample dried on the solid surface returned significantly the least amount of conidia, as it yielded 3.12 × 10⁷ conidia with a loss ratio of 36.21%.

Fig. 4

Count of conidia obtained from 1 g fresh sample or its equivalent weights of dried samples

Biological activity of the resulting conidia

A bioassay was carried out to assess the efficacy of the resulting conidia against the red spider mite. The final result of this bioassay comprehensively expresses the vitality, germinability, and pathogenicity of the conidia before and after subjection to drying methods. The biological activity of the obtained conidia was assessed by applying a similar concentration of conidia from each sample, which was assumed to kill 50% of T. urticae mites. Looking at the results represented in Fig. 5, considering the calculated LSD value of 9.2, it could be seen that the fresh, condensing water vapor, and desiccant samples revealed almost similar mortality percentages of 53.95, 51.28, and 52.56%, respectively. When the sample was dried on a net, the mortality rate decreased significantly to 42.31%, while drying the sample on a solid surface resulted in the lowest mortality rate of 32.05%, which was significantly lower than all other samples.

Fig. 5

Mortality percentages after treating T. urticae with LC₅₀ concentration of conidia obtained from different samples

Discussion

Traditionally, in most cases, drying of conidiated culture accomplished through either a simple method such as air drying or using other techniques, which are usually complicated. Air drying was known to be a time-consuming process (Mathulwe et al. 2022). Also, it caused a reduction in both spore count and pathogenicity, as demonstrated in this study. On the other hand, alternative techniques are often complex, e.g., Moslim et al. (2005) detached spores from the media by shaking them with a tween 80 solution. Subsequently, a separation machine was utilized to segregate the solid substrate, and the spores were then collected using a manifold vacuum filtration machine before undergoing a drying process. Also, Pham et al. (2010) performed an initial drying process, followed by sieving the spores and subsequently subjecting them to vacuum drying. This last technique had the advantage of achieving a moisture content of less than 5%, whereas the previous method resulted in a higher spore yield. Nevertheless, both techniques required specific equipment, which may not be readily available to low-technology producers. The present study presented simple techniques appropriate for low-tech producers, as these techniques are based on providing low relative humidity at low temperatures. The two methods examined in this study, namely condensing water vapor and desiccant techniques, decreased the moisture level to approximately 12–13%, a level close to the 9% threshold recommended by Sala et al. (2019) for enhancing the shelf life of conidia.

Condensing vapor technique is a promising candidate for widespread application in a room filled with net racks and provided with a fan, electric heater, and cold air conditioner that works to condense water vapor and get rid of it outside the room through the air conditioner’s drainage system. As explained above, in the case of the incubator, the electric heater drives the air conditioner to operate more frequently, resulting in a higher condensation of vapors and a decrease in the relative humidity in the room.

From the principles of physics, it is known that the rate of evaporation is reduced in regions with high relative humidity, so this gives an advantage to condensing vapor and desiccant techniques in areas characterized by such conditions, as these techniques operate autonomously in isolation from the natural air.

Conclusion

The condensing water vapor and the desiccant techniques showed superiority and revealed similar results regarding drying rate, final moisture content, conidia yield, and pathogenicity. Air drying on a net technique needed additional time and left a higher final moisture content. Although the number of conidia yielded from this technique was almost similar to the aforementioned two techniques, a significant decrease in pathogenicity was observed. Air drying on a solid surface technique was the least efficient regarding final moisture content, conidia yield, and pathogenicity. Drying on a net may provide a reasonable technique if there is a shortage of equipment. Anyway, producers must eliminate drying on a solid surface.