Brown rot is a common fungal disease in the Southeastern United States that affects stone fruits like peaches, cherries, and plums. Monilinia fructicola is a tan, fuzzy fungus that grows on tree flowers, twigs, and fruit, and is the most common cause of brown rot. Once brown rot infects a tree, the damage is irreversible, causing significant crop losses in commercial orchards. Farmers try to control the spread of M. fructicola by applying chemical sprays, called fungicides, to fruit trees in the spring.

In M. fructicola, a biosynthesis gene named MfCYP51 encodes a major protein critical to the fungi’s survival. Certain fungicides, called demethylation inhibitor (DMI) fungicides, stop M. fructicola from growing by targeting this protein. Over the last 20 years, DMI fungicides have become less effective against brown rot as M. fructicola becomes more resistant. 

Living microorganisms and their metabolites can control fungal diseases as an alternative to chemical fungicides, known as biofungicides. Many bacteria produce compounds like hydrogen cyanide and pyrrolnitrin that damage fungal cells. However, scientists have not found a biofungicide suitable for commercial production.​ A group of researchers at Clemson University in the USA and Huazhong Agricultural University in China set out to test whether the soil bacteria Pseudomonas chlororaphis and Bacillus subtilis could be effective biofungicides against brown rot. 

The researchers performed an incubation experiment to analyze how the fungal cells make their critical protein from the MfCYP51 gene, a process called gene expression, under different fungicide treatments. They used 3 M. fructicola strains sensitive to traditional DMI fungicides and 3 resistant strains. They tested these with 5 different fungicide treatments: a DMI fungicide, a mixture of P. chlororaphis metabolites, a mixture of living B. subtilis cells, a DMI fungicide + P. chlororaphis metabolites, and a DMI fungicide +  B. subtilis cells. They also set up a control treatment with sterile water instead of fungicide.  

After 6 hours, the researchers extracted the molecule that measures gene expression, called RNA, from the fungi. They found that both the P. chlororaphis treatment and the DMI fungicide + P. chlororaphis treatment substantially reduced MfCYP51 expression in both the sensitive and resistant isolates compared to the controls. In contrast, the DMI fungicide, B. subtilis, and DMI fungicide + B. subtilis treatments increased MfCYP51 expression in the resistant isolates. The researchers concluded that P. chlororaphis has a unique ability to reduce gene expression, unlike other biofungicides.

​To further investigate how the biofungicide treatments work, the team tested whether P. chlororaphis and B. subtilis produced the anti-fungal metabolite pyrrolnitrin. They added 1 milliliter of each treatment to a machine that separates and identifies liquid compounds using high pressure, called a high-performance liquid chromatograph. They found that the P. chlororaphis treatment contained pyrrolnitrin, so they hypothesized that it was responsible for reducing gene expression.

Additionally, the researchers tested their 5 treatments on brown rot in fruit. For each treatment, they took 10 Gala apples, washed and sterilized their surfaces, and sprayed them with the fungicides. After 24 hours, they poked holes in the apples with toothpicks and added 20 microliters of liquid containing M. fructicola cells to the poked holes. They placed the apples in a humid chamber for 5 days and measured the number and size of brown rot spots that developed on their surfaces. 

Despite decreasing MfCYP51 expression, ​the researchers found that P. chlororaphis treatment alone didn’t reduce brown rot disease compared to the control, but the DMI fungicide and B. subtilis treatments did. The mixed treatments of DMI fungicide + P. chlororaphis and DMI fungicide + B. subtilis both reduced brown rot disease compared to the control, but only the DMI fungicide + P. chlororaphis treatment prevented it. 

​The researchers concluded that although biofungicides may not be fully effective on their own, commercializing them could allow farmers to use less DMI fungicide. They explained that scaling back the use of DMI fungicides would slow M. fructicola resistance. Finally, they proposed that future researchers test biofungicide mixtures containing pyrrolnitrin in the field to observe real-world effects on stone fruit trees.

The post Using bacteria to fight fungal brown rot appeared first on Sciworthy.