Detecting Fungal Infections in Almonds
Manually select almond kernel samples that are pasteurized and shelled.
As mentioned in the 'Before Start Instructions', the almond kernels should be collected from multiple harvest cycles.
These particular samples were obtained from the 2012 and 2013 almond seasons.
The samples should be shelled and should not have any type of physical damage or any marks.
Record weight and size of almond kernels Separate the samples into two groups.
Out of the 550 almond kernels collected 460 of those will be infected with two types of fungi that commonly affect almond crops.
230 will be infected with A. flavus and another 230 will be infected with A. parasiticus.
The remaining 90 kernels will remain uninfected and will serve as a control group.
Obtain A. flavus and A. parasiticus isolates to infect almonds.
These two types of fungi were chosen due to their well known colonization on the surface of almond kernels.
Prior to inoculation, take the fungi isolates and grow them in culture.
Use 5% reconstituted tomato juice agar, in this case it will be V8 juice agar, and grow each isolate for 6 days at a temperature of 31 °C in a dark environment.
Once fungal isolates have been grown, conidia is collected using a sterilized cotton swab and is deposited in sterile deionized water.
A haemocytometer will be used to quantify the water solution with conidia and it will then be diluted to a concentration of 105 spores per ml-1.
Create incision on the bottom right of almond kernels using a B-D® 16G1 Precision Glide® Needle.
This incision serves as the point of infection where the conidia in water would be placed.
After making the incision on the surface of the almond, the kernels will be sterilized by submerging them in 10% bleach solution for a period of 7 minutes.
The kernels will then be rinsed three times with sterile deionized water and will be submerged a second time into a Dichloran solution for 2 seconds.
Remove moisture from surface of almonds by blotting them and move them to a biological safety cabinet for a period of 10 to 15 minutes.
Inoculate each almond with 5 μl of the fungi solution.
As mentioned earlier, almonds will be in two separate groups: infected and control.
The infected almonds will be inoculated with either A. flavus or A. parasiticus.
Once the almonds have been inoculated, add deionized water to the container where they are located and incubate them for a period of 7 days at a temperature of 31° in a dark place.
Once the incubation period is over, wash almonds with deionized water to remove any traces of fungi spores from the surface.
Place almond kernels in a fume hood for a period of 12 hours.
Once the 12 hours are up keep kernels at a constant temperature of 2.6°C.
Take almond and put it under the spectrophotometer, then place black optic mask in the space between these two.
Scan the surface of the almond kernel using the spectrophotometer, for each individual almond kernel take 32 scans of the face across a wavelength range of 400 nm to 2500 nm, in 2-nm increments.
Use statistical analysis software to analyze the data gathered from all three groupings of almonds.
