E L NIVEL VULGAR
II. Hablas generadas por el lugar de residencia.
In general, a higher number of dead and mycotic larvae were found with the higher water content in soil, as expected, demonstrating the importance of moisture for conidia germination and fungal infection (p<0.01; Figure 2.10). Mycosis also increased with the concentration of the conidial suspension, with lower percentage of sporulating supporting cadavers at 105 conidia/mL than at 107 and 109 (p<0.01), although differences in mycosis at these two higher concentrations were not always significant.
Figure 2.10 Bioassays of Costelytra giveni with entomopathogenic fungi. A. Larve with mycelial growth and sporulation. B. Dead larvae with or without signs of fungal infection were transferred to individual wells and kept at 22°C for at least 40 days to determine mycosis.
2.3.7..1 Mortality and mycosis caused by Metarhizium spp. and Beauveria bassiana in 20% (w/v)
soil moisture
At the lowest conidia concentration (1 x 105 conidia/mL), inoculation of soil with the M. guizhouense BK41 was the first to reach 33% mortality of grass grub larvae, after 15 days post inoculation (DPI) (Table 2.10). By day 20, at this conidia concentration, most isolates presented larval mortalities between 15 and 40% (Figure 2.11). The isolate M. guizhouense F16 was the first to cause a mycosis of
25% after 20 DPI (LSD = 11.9; Figure 2.11) while M. robertsii F447 was the only isolate to have a value close to 30% by the end of the study (p<0.01; Figure 2.12).
Table 2.10. Costelytra giveni mortality, in 2nd and 3rd instar larvae, at different conidia
concentrations in 20% (w/v) soil moisture over 40 days. All isolates belonged to different species of Metarhizium except for Beauveria bassiana Bb21.
1 x 105 (cond/mL) 5 10 15 20 25 30 35 40 A1080 5.5 5.5 11.1 19.5 30.6 63.9 80.5 88.9 Bb21 8.3 16.7 25 25 33.3 83.3 83.3 83.3 Bk41 8.3 25 33.3 41.7 50 50 50 75 F16 8.3 16.7 16.7 25 50 75 75 83.3 F447 0 8.3 8.3 8.3 33.3 50 75 100 F672 0 0 0 0 25 58.3 66.7 75 F99 0 0 0 16.7 41.7 58.3 58.3 83.3 LSD 14.5 20.0 18.9 18.8 24.4 23.3 20.3 16.0 1x107 (cond/mL) 5 10 15 20 25 30 35 40 A1080 0 0 5.5 8.3 41.7 83.3 91.7 94.5 Bb21 0 0 0 8.3 25 58.3 58.3 75 Bk41 0 8.3 8.3 16.7 25 75 91.7 100 F16 8.3 16.7 16.7 41.7 50 83.3 91.7 100 F447 0 0 16.7 16.7 33.3 83.3 100 - F672 0 16.7 16.7 16.7 41.7 91.7 100 - F99 25 41.7 58.3 58.3 75 91.7 100 - LSD 14.5 20.0 18.9 18.8 24.4 23.3 20.3 16.0 1x109 (cond/mL) 5 10 15 20 25 30 35 40 A1080 2.8 2.8 8.3 8.3 55.5 88.9 94.5 100 Bb21 0 8.3 8.3 8.3 50 83.3 83.3 91.7 Bk41 0 8.3 8.3 16.7 50 75 100 - F16 8.3 25 25 50 66.7 100 - - F447 0 8.3 33.3 33.3 50 83.3 100 - F672 0 25 25 33.3 66.7 66.7 100 - F99 0 8.3 8.3 25 58.3 83.3 91.7 100 LSD 14.5 20.0 18.9 18.8 24.4 23.3 20.3 16.0 Controls 3.3 6.7 6.7 8.3 11.7 11.7 11.7 11.7 A B
At a conidia concentration of 107 conidia/mL, M. novozealandicum F99 had the highest larval mortality of 42% after 10 DPI (p<0.01) which increased to 60% after 20 DPI (Table 2.10). Fungal colonisation and sporulation on the dead larvae was detected at significant levels only after 30 days from inoculation (p<0.01). After 35 DPI, 25% of cadavers in the M. guizhouense Bk41 treatment supported fungal colonization, while 58% and 75% of cadavers were with mycosis for the M. anisopliae F672 and M.
robertsii F447 treatments, respectively (LSD = 17.6; Figure 2.12).
Figure 2.11 Costelytra giveni survival, in 2nd and 3rd instar larvae, after 20 days of inoculation with a
conidial suspension and at 20% w/w humidity content. Total larvae population in every treatment was evaluated as percentage of alive, dead without mycosis or dead with mycosis. Therefore, total dead is “dead” + “mycosis”. A = 1 x 105 conidia/mL; B = 1 x 107 conidia/mL; C = 1 x 109 conidia/mL. p-values: Alive (p <0.01); Dead (p<0.01); Mycosis (p=0.281). Control dead larva = 8.3%
At 20% w/w moisture content and 20 DPI, larval survival was highest with the M. anisopliae F672 treatment at 105 conidia/mL, while the lowest survival was in the M. novozealandicum F99 at 107 conidia/mL (p<0.01). Mortality of C. giveni larvae with the M. novozealandicum F99 treatment at 1x107 conidia/mL was higher than the M. anisopliae A1080 treatment at all conidial suspensions, and to M.
robertsii F477 at conidial suspension 105 and 107 conidia/mL (p<0.01). Mycosis for B. bassiana Bb21 was below 1% at 105 conidia/mL while no cadavers with signs of fungal infection were observed at the two other higher conidia concentrations.
Figure 2.12 Costelytra giveni survival, in 2nd and 3rd instar larvae, after 35 days of inoculation with a
conidial suspension and at 20% w/w humidity content. Total larvae population in every treatment was evaluated as percentage of alive, dead without mycosis or dead with mycosis. Therefore, total dead is “dead” + “mycosis”. A = 1 x 105 conidia/mL; B = 1 x 107 conidia/mL; C = 1 x 109 conidia/mL. p-values: Alive (p <0.01); Dead (p<0.075); Mycosis (p<0.01). Control dead larva = 11.7%
At the highest conidia concentration (109 conidia/mL) M. guizhouense F16 and M. anisopliae F672 treatment resulted in mortalities of 25% after 15 DPI while in M. robertsii F447 had the highest mortality with 33% (Table 2.10). Mycosis signs were first detected in the M. guizhouense F16 treatment, representing 17% after 20 DPI (LSD = 16.7) and 42% after 30 DPI (LSD = 17.6; Figure 2.11). At the end of the evaluation, 40 DPI, more fungal infected cadavers were seen in the M. robertsii F447 treatment (67%), followed by treatments with M. guizhouense Bk41 and F16 with 42% (p<0.01).
After 35 DPI, the highest values of C. giveni larvae alive were found in the M. guizhouense Bk41 and
M. novozealandicum F99 at 105 conidia/mL, while no surviving larvae were found in M. anisopliae F672 and M. robertsii F447 treatments at 107 and 109 conidia/mL or with M. novozealandicum F99 at 107 conidia/mL and M. guizhouense Bk41 at 109 conidia/mL (p<0.01). The highest mycosis percentages were found in M. anisopliae F672 and M. robertsii at 107 conidia/mL (p<0.01).
2.3.7..2 Mortality and mycosis due to Metarhizium spp. and Beauveria bassiana in 30% (w/v) soil
moisture
The increase in moisture helped to accelerate the infection process for all the conidia concentration tested. At 105 conidia/mL, the treatment M. guizhouense F16 was the first to reach 50% mortality after 5 days post inoculation (Table 2.11). By 15 DPI isolates M. anisopliae A1080 and M. guizhouense F16 both had mortalities of 75%(LSD 5.2; Figure 2.13). The treatment M. guizhouense F16 was again the first isolate to reach a 25% mycosis of total larvae treated but, in this case, just after 10 DPI (p<0.01; LSD = 2.0). However, as previously noticed, no remaining cadavers showed mycosis for this isolate. At the end of the study, 35 DPI, the highest mycotic values were determined for isolates M. robertsii F447 (42%) and M. guizhouense BK41 (50%) (p<0.01; LSD = 2.2). B. bassiana Bb21 and M. anisopliae F672 were the only two treatments where no larvae with signs of mycosis were found (Figure 2.14).
At the intermediate conidia concentration of 107 conidia/mL, the treatment M. guizhouense BK41 was the first to kill all larvae just after 5 DPI (Table 2.11; p<0.01). The next treatment to kill all the larvae after 15 DPI was M. anisopliae F672 which was followed closely by M. anisopliae A1080 with 92% (p<0.01). Also, mycoses on the dead larvae were first detected at significant levels in M. anisopliae A1080 (25%) after just 5 days from inoculation (p<0.05). At 20 DPI isolates which had mycosis above 25% were M. robertsii F447, M. anisopliae A1080 and F672, and M. guizhouense F16 with 66.7% (Figure 2.13). This treatment, at the end of the study, was the only one to reach 83% of cadavers supporting sporulation followed by M. anisopliae A1080 and F672 with 42% and 50% respectively (p<0.01; Figure 2.14).
Table 2.11. Costelytra giveni mortality, in 2nd and 3rd instar larvae, at different conidia
concentrations in 30% (w/v) soil moisture. All isolates belonged to different species of
Metarhizium except for Beauveria bassiana Bb21. Larval survival was monitored every 5
days after inoculation.
1x105 (cond/mL) 5 10 15 20 25 30 35 A1080 33.3 55.5 75 100 - - - Bb21 0 0 0 0 25 25 50 Bk41 25 50 50 75 75 100 - F16 50 50 75 75 100 - - F447 25 25 50 75 100 - - F672 0 0 16.7 50 58.3 58.3 66.7 F99 25 25 25 25 50 58.3 100 LSD 23.1 18.4 17.8 5.2 9.2 12.6 9.2 1x107 (cond/mL) 5 10 15 20 25 30 35 A1080 83.3 83.3 91.7 100 - - - Bb21 83.3 83.3 83.3 100 - - - Bk41 100 - - - - F16 58.3 66.7 75 100 - - - F447 50 50 58.3 100 - - - F672 50 75 100 - - - - F99 66.7 66.7 75 75 83.3 83.3 83.3 LSD 23.1 18.4 17.8 5.2 9.2 12.6 9.2 1x109 (cond/mL) 5 10 15 20 25 30 35 A1080 38.9 52.8 69.5 91.7 100 - - Bb21 8.3 25 50 66.7 66.7 75 83.3 Bk41 25 75 100 - - - - F16 41.7 58.3 75 100 - - - F447 25 58.3 91.7 100 - - - F672 25 33.3 66.7 100 - - - F99 33.3 58.3 83.3 100 - - - LSD 23.1 18.4 17.8 5.2 9.2 12.6 9.2 Controls 6.7 6.7 10.0 10.0 11.7 13.3 13.3
At a concentration of 109 conidia/mL the treatment M. guizhouense BK41 after 10 days since inoculation was the first to 75% of larvae dead (Table 2.11). After 20 DPI almost all Metarhizium strains
At 30% w/w moisture content and at 20 DPI, the highest larvae mortality was found in M.
novozealandicum F99 at 109 conidia/mL (p<0.01), while mycosis was highest for M. robertsii F447 and
M. guizhouense Bk41 at 109 conidia/mL, and for M. guizhouense F16 at 107 conidia/mL (p<0.01). After 35 DPI, alive larvae were only found in treatments M. anisopliae F672 and B. bassiana Bb21 at 105 conidia/mL (p<0.01), while the highest values of larvae with mycosis were found in M. anisopliae F672 and M. robertsii F447 both at 109 conidia/mL and in M. guizhouense F16 at 107 conidia/mL (p<0.01).
Figure 2.13 Costelytra giveni survival after 20 days of inoculation with a conidial suspension and at 30% w/w humidity content. Total larvae population in every treatment was evaluated as percentage of alive, dead without mycosis or dead with mycosis. Therefore, total dead is “dead” + “mycosis”. A = 1 x 105 conidia/mL; B = 1 x 107 conidia/mL; C = 1 x 109 conidia/mL. p-values: Alive (p <0.01); Dead (p<0.01); Mycosis (p=0.01).
Signs of fungal infection, were detected in 33% of larvae after 10 DPI at 109 conidia/mL in M.
guizhouense BK41 and M. robertsii F447. Both isolates increased mycosis on larvae to 58% after 20
DPI and were followed close by treatment M. guizhouense F16 with 42% (p<0.01; Figure 2.13). At the last evaluation day, the highest mycotic values were recorded for M. robertsii F447 and M. anisopliae F672 with 66.7%, while for M. guizhouense BK41 it was 58% (p<0.01; Figure 2.14). At this concentration, no larvae with signs of mycosis were found in the treatment with B. bassiana Bb21.
Figure 2.14 Costelytra giveni survival after 35 days of inoculation with a conidial suspension and at 30% w/w humidity content. Total larvae population in every treatment was evaluated as percentage of alive, dead without mycosis or dead with mycosis. Therefore, total dead is “dead” + “mycosis”. A = 1 x 105 conidia/mL; B = 1 x 107 conidia/mL; C = 1 x 109 conidia/mL. p-values: Alive (p <0.01); Dead (p<0.367); Mycosis (p=0.01).