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Nitrogen is an essential building block of proteins, amino acids, and DNA. Without nitrogen, cells can’t function or reproduce. Nitrogen is abundant in the atmosphere as dinitrogen gas, but plants and animals can’t access atmospheric nitrogen. Instead, we rely on biologically available forms like ammonia and nitrate. 

Specialized microbes can transform atmospheric nitrogen into ammonia via a process called biological nitrogen fixation. Nitrogen-fixing soil bacteria have a unique protein complex, the nitrogenase complex, that facilitates this nitrogen transformation. The nitrogenase complex is activated by a group of genes known as nif genes. 

Legume plants like peanuts, peas, and beans have nitrogen-fixing bacteria in the soil zone around their roots, called the rhizosphere. The symbiotic relationship between nitrogen-fixing soil bacteria and legume plants supplies most of the biologically available nitrogen to life on Earth.

However, one piece of the puzzle remains unexplored: the role of soil viruses in these nitrogen transformations, referred to as the nitrogen cycle. Viruses are microscopic particles made up of genetic material and proteins, but they can’t reproduce without a host cell. Scientists consider viruses to be biological entities that are halfway between living and non-living.

To reproduce, viruses contact a host cell and inject their genetic material into it. The virus’s genetic material takes over the host cell’s genome and forces the cell to produce proteins that assemble into new viruses. As new viruses are created, they can incorporate genes from the host cell into their own genetic material. Genes that are transferred from a host cell to a virus are known as auxiliary metabolic genes (AMGs). Viruses can carry AMGs and pass them to other hosts or use them to increase their own ecological fitness. 

Scientists from China, Spain, and the Czech Republic set out to investigate whether viruses from legume roots could increase local soil nitrogen fixation by expressing nif AMGs or by transferring nif genes to other bacteria. First, they determined the global distribution of viral nitrogen-fixing genes by analyzing about 8.6 million viral genomes from the Integrated Microbial Genomes/Virus database. They looked for the DNA sequences of known nitrogen-fixing genes and the locations where the viruses were sampled. They found that only 0.003% of the viruses in the database had at least 1 nitrogen-fixing gene, but they clustered in the same locations as nitrogen-fixing bacteria. 

The team also found 3 types of viruses that most commonly carried nitrogen-fixing genes: Kyanoviridae, Nudiviridae, and Bronfenbrennervirinae. Kyanoviridae viruses carried a nif gene called nifU. The researchers used a computer program called DRAM-v to confirm that nifU was a fully functional gene and a real tool that viruses could use. 

Next, the team collected soil samples from a cowpea field in Nanjing, China, to test whether the cowpea roots influenced the abundance of viruses using nitrogen-fixing genes. They collected soil from the cowpea rhizospheres and soil away from the plants. They analyzed both viral and bacterial RNA in the soil samples to identify active genes, using a sequencing process called metatranscriptomics. They found that the viral nifU gene was actively expressed in the cowpea rhizosphere samples more than in the non-crop soil. Although about 96% of nifU genes were being expressed by bacteria, viruses contributed about 4%. 

The researchers then assessed whether rhizospheric viruses changed the amount of nitrogen being fixed in these soils. They set up small, enclosed containers of sterile soil and added either bacteria or a mixture of bacteria and viruses extracted from the cowpea rhizospheres. They found that soil with viruses had higher total nitrogen (36 milligrams per kilogram, or mg/kg) than soil without viruses (17 mg/kg). 

Finally, they adjusted the air in each container to have a different form of dinitrogen gas. Atoms of the same element that have different atomic masses are known as isotopes. Nitrogen has 2 stable isotopes: lighter nitrogen-14 and heavier nitrogen-15. Bacteria or viruses that fix nitrogen at natural isotope abundances will take up nitrogen-14. But scientists can track who in the population fixes nitrogen by adding nitrogen-15 to the air, a process called nitrogen-15 stable isotope probing. Nitrogen-fixers will incorporate the heavy nitrogen-15 into their biomass over time and increase their weight, allowing researchers to separate and identify them.

The team grew the bacteria and viruses with nitrogen-15 for 35 days, then used stable isotope probing to separate the nitrogen fixers. They detected viral nifU AMGs among the heavier biomass, confirming that rhizospheric viruses were fixing nitrogen. 

The team concluded that rhizospheric viruses and the viral nifU AMG can increase nitrogen fixation in cowpea soils. The team suggested that although viruses with nitrogen-fixing genes are rare, they can influence the soil nitrogen cycle. They recommended that future researchers further test the contribution of viral nifU genes to plant nitrogen fixation with controlled infection experiments.

The post Viruses help plants obtain nitrogen appeared first on Sciworthy.

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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.