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 .
