Relaciones laborales

6.5.1

Background

The propriety broth for fermentation of B. bassiana K4B3 had always been adjusted at pH 5.5 at the start of production. The effect of start pH of the medium on the production of insecticidal metabolites was unknown for this fungus. Previous attempts to fix pH at 5.5 throughout the growth period resulted in poor products. The pH of the end product (three or four days old) which delivers the highest insecticidal ability was in the range of 3.8 to 4. No attempt was made to check if assisting the fungus to grow at lower or higher start pH would expedite the production of the insecticidal metabolites. The general pH of insect surfaces and haemolymph are at 7.7 or pH 6.8 respectively (Chapman et al., 2013; St. Leger et al., 1998). Thus the insecticidal activities were compared using supernatants of K4B3 cultured in media with different start pH.

6.5.2

Materials and methods

pH and time course study

A series of submerged K4B3 cultures with start pH of 3, 5, 7 and 8 (adjusted using 1 M hydrochloride acid HCl and 1 M NaOH) were evaluated for insecticidal ability. The glucose-based media were prepared according to the ingredients in Table 6.1. A total of 2% (2 ml) of the final volume (100 ml) of a three-day-old fermentation broth of B. bassiana K4B3 was used to inoculate each flask. The flasks

were constantly shaken at 225 rpm to provide more turbulence and incubated at 27 °C for seven days,

sampled every 24 h. The samples were centrifuged at 4000 rpm for 15 min to separate mycelia and supernatants. The supernatants were assayed against aphids and mortality observed after 24 h.

30 63 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 M or ta lity (% ) Day pH5 pH8

Repeat experiment using pH 5.5 and above

Based on the results from the time course study on pH, the same experiment was repeated with start pH ranging from 5.5 to 9.5. The pH of the media before and after inoculation, and after three days fermentation was recorded. The inoculum was 2% with pH of 3.8.

6.5.3

Results

pH and time course study

The direct toxicity to aphids of supernatants sampled over seven days was assessed. All the supernatants had some toxicity to aphids, compared to the controls (blank medium), after topical application. Cadavers of treated aphids appeared ‘desiccated’ and ‘flattened’ with outstretched legs and proboscis still embedded in the leaf. The mortality of aphids in the control was below 1%. Altering the start pH of the medium above pH 5.5 did not alter the toxicity of the supernatants significantly. Following spray application, the mortality of aphids Myzus persicae at 24 h was the highest using supernatant sampled after three days of growth. The insecticidal activity was reduced using supernatant sampled on the sixth day but increased again using supernatant from the seventh day (Figure 6.5). The pH of the unbuffered glucose-based cultures generally decreased to around pH 3.7 after three days (Figure 6.6). Attempts to improve fungal production using a start pH 3 not only slowed the growth of the culture, but the resulting bioactivity was reduced for all days. The mortality values from three-day-old supernatants with start pH 7 and pH 8 were significantly higher than the mortality values for supernatants with start pH 3 and pH 5 (p<0.05).

Figure 6.5. Mortality of aphids Myzus persicae 24 h after treatment with supernatants from glucose- based cultures with different start pH. LSD for comparing different sampling days at same pH is 29.9 and LSD for all other comparisons is 31.0.

The pH of all cultivation decreased over time. Acidic products were produced and the pH remained between 3.5 and 4.0 (Figure 6.6). The colour of the culture differed when grown at different start pH (Figure 6.7-6.8). K4B3 was growing exponentially and plateaued by the seventh day (Figure 6.9). Bibasic phosphate salt (potassium diphosphate) is a high buffering agent (Biomedicals, 2014 ) but it did not buffer the pH of the broth.

Figure 6.6. Changes in pH over seven days’ incubation of supernatant samples, from media started at different pH.

Figure 6.7. Three-day-old glucose-based cultures of K4B3 grown at different start pH. Left to right; start pH 3, 5, 7 and 8.

Figure 6.9. Dry weight (g) of mycelial samples for seven days from a glucose-based culture of K4B3

with start pH 8 and fermentation at 27 °C. Values are mean ± standard deviations.

Effect of pH 5.5 and above

A second experiment was used to investigate effect of pH 5.5-9.5 as start pH of medium. After 18 h, colour change was observed in K4B3 cultures grown with start pH above 5.5. The range of pH tested showed growing K4B3 with start pH above 5.5 resulted in achieving insecticidal supernatants (Table 6.2 and Figure 6.10), even medium with start pH 9.5 which was cloudy when inoculated.

Table 6.2. pH values of media before inoculation, after inoculation (with 2 ml of culture) and after three

days of fermentation at 27 °C.

Start pH of media pH directly after inoculation pH of supernatant on 3rd _day

5.5 5.43 3.43 6.5 6.32 3.45 7.5 6.92 3.55 8.1 7.34 3.40 8.5 7.66 3.56 9.5 8.62 cloudy 3.54

Figure 6.10. Mortality of aphids Myzus persicae after 24 h treatment using supernatants of three- day-old K4B3 culture with media start pH above 5.5. Values are mean ± standard deviations. N=30 for each treatment. C=control.