Aphids, diamondback moth (DBM) larvae, brine shrimps, mosquito larvae, thrips, armyworm larvae and phytopathogenic microbes were selected for bioassays based on their economic importance, ease to culture and availability. The materials used for bioassay were as below.
One liter of K4B3 broth was produced using ingredients and culture parameters (as listed in Appendix
A.1.1 and A.1.4). Approximately 900 ml of the broth was centrifuged at 4000 rpm at 20 oC for 15 min
to separate the mycelia and supernatant. Five hundred ml of supernatant was separated from mycelia through centrifugation and passed through a 0.2 µm filter (Sartorious). A sample was used directly for bioassays without organic solvent treatments. Three hundred ml of the cell-free supernatant and the mycelia (170 g wet weight) were freeze dried (using a Thermo modulyo unit connected to a vacuum pump Savant VLP120) for 24 h. The freeze-dried mycelial material was pulverized using a blender and shaken three times in 200 ml methanol (MeOH) for 15 min. Fresh MeOH was used for each of the extraction. Extracts were filtered through a Buchner funnel to separate the mycelia and the filtrate. The MeOH filtrates were pooled and the solvent was evaporated using a rotary evaporator (Buchi).
When dried, the mycelial extract weighed about 9 g. All materials were kept at -20 °C prior to the
bioassays. A series of concentrations was prepared in 0.1% Tween 80 aqueous solution using the crude extract and untreated cell-free supernatant to determine the lethal concentration which kills 50% of
the test subject population (LC50). Mortality was monitored after 24 h. The LC50 value was obtained
from reverse log of the estimated value from the curve. Negative control was 0.1% Tween 80 containing growth medium.
3.2.3
Bioassays
Aphid (Myzus persicae) and DBM caterpillar (Plutella xylostella) assays
Please refer to the approach described in 2.2.
Unfiltered supernatant and DBM (Plutella xylostella)
All filtered supernatants (from 3.2.2) with 0.1% Tween 80 added were tested on ten first and second instar DBM caterpillars. Ten eggs and pupae in triplicate treatments were also immersed in the supernatants for 15 s and left to dry. Unfiltered supernatant (without mycelia obtained by passing the broth through cotton swab) to which 0.1% Tween 80 was added and tested on ten DBM caterpillars. The amount of blastospores present in the unfiltered material was counted using a haemocytometer (Hawksley: Improved Neubauer). Control was blank medium with 0.1% Tween 80 added. All treatments were in triplicates and the caterpillars were offered leaf discs of cabbage after treatment.
The caterpillars were incubated at 22 °C, 80% humidity control and 16 h of light and 8 h of dark. They
Brine shrimp (Artemia salina) assay
K4B3 culture was grown for seven days at Lincoln University as described in Chapter 2. Two ml samples
were collected on a daily basis and stored at -20 °C. When all samples were available, they were
thawed and centrifuged to separate the mycelia and supernatant at 4000 rpm for 15 min. The separated mycelial samples were then extracted with 1 ml MeOH by vigorously shaking the suspension for 10 min. They were then centrifuged at 4000 rpm for 10 min and the MeOH extract was retrieved and dried. The extracts were weighed and re-suspended in 0.5 ml saltwater added with 0.1% dimethyl sulfoxide (DMSO) (Hartl and Humpf, 2000; Molina-salinas and Said-Fernandez, 2006; Solis et al., 1993). One hundred µl aliquots of all daily mycelial suspensions and supernatants were added into wells of a 96-well clear flat bottom polypropylene micro-titer plate with three replicates per sample. Brine shrimps, Artemia salina, (purchased from a Christchurch petshop) were hatched from eggs in 3% salt water within 24 h. Ten newly hatched brine shrimps were placed in each well of a 96-well micro- titer plate consisting of 100 µl of salt water mixed with 100 µl of the mycelial extract and supernatant.
The shrimps were incubated at 22 °C in constant light. Mortality was observed after 1 h, 6 h, and 24 h.
The negative control was sea water with 0.1% DMSO. The positive control was potassium dichromate
K2Cr2O7 (0.4 mg ml-1 salt water) and pyrethrum (5 ml l-1) (Molina-salinas and Said-Fernandez, 2006).
The concentration-response data was transformed into a straight line by means of logarithm
transformation of the concentration and the LC50 was estimated. The actual value was then obtained
by reverse log of the LC50 value estimated from the curve (Hartl and Humpf, 2000; Meyer et al., 1982).
Mosquito larvae assay
Mosquito larvae, Culex pervigilans, were collected from ponds in the Canterbury area (New Zealand) for bioassay. Five ml of the daily supernatant samples (filtered) from 2.1.2 were tested against ten larvae respectively in a 6-well flat bottom tissue culture plate. Six different concentrations of mycelial crude extract from 3.2.2 (in 5 ml 0.1% Tween 80 aqueous solution) were also tested. A series of supernatant from three-day-old culture (from 2.1.2) at 10, 30 and 50% concentration was also tested
directly. The larvae were incubated at 22 °C in the dark. Ten larvae placed in each well were monitored
Thrips assay
Nymphs of thrips, Frankliniella occidentalis, were collected from a cabbage nursery at Lincoln University. Ten nymphs were sprayed with K4B3 material from 3.2.2 using the same approach as
described in 2.2 for aphid assay. The nymphs were incubated at 22 °C, 80% humidity control and 16 h
of light and 8 h of dark. The nymphs were monitored for mortality after 24 h.
Anti-feedant assay
Eggs of armyworm, Spodoptera litura, were obtained from the New Zealand Institute for Plant & Food Research Limited in Auckland. Immediately following hatching, the neonate first instar larvae were
used. Three sets of freeze dried supernatant material (from 3.2.2) weighing 0.3, 3 and 30 mg,
respectively, were mixed into 300 mg portions of noctuid wheat germ based diet. The artificial diet
was offered to ten Spodoptera litura first instar larvae. The larvae were incubated at 22 °C, 80%
humidity control and 16 h of light and 8 h of dark. Mortality was recorded after 24 and 48 h.
Disc diffusion assay
Centrifuged supernatant samples collected over a duration of seven days (see 3.2.2) were used to test against several bacteria and fungal species. Fifty µl of the supernatant was applied onto a paper disc (8 mm diameter Advantec) and dried. The paper disc was placed on agar which was then plated with
the test microbes and left overnight at 25 °C. The size of the clear halo around the paper disc was
measured. Cycloheximide (1 mg ml-1, Sigma-Aldrich) and chloramphenicol (0.1 mg ml-1, Sigma-Aldrich)
were used as positive controls. Plugs of mycelia from fungi to be tested were placed adjacent to the paper discs treated with crude supernatant on PDA plates and observed for inhibition zones. The radius of the inhibition zones were measured using a ruler. Freeze-dried supernatant materials (from 3.2.2) at 100,000 ppm and MeOH extracts of K4B3 mycelium at 50,000 ppm were tested as well for antimicrobial activities.