Bacterial Consortia Shield Sugar Beets from Root Disease

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Cultivated (left) vs. Wild (right) Sugar Beets: Image by Peggy Greb, courtesy USDA

Life for a sugar beet isn’t as sweet as one would think. For starters, it’s prey to the notorious fungal predator, Rhizoctonia solani, which also causes great harm to potatoes and rice.

But sugar beets have a trick or two up their leaves. One particularly impressive maneuver has just been reported by Jos M. Raaijmakers at Wageningen University in Wageningen, the Netherlands and a team of research scientists from around the world. It appears that sugar beets defend their roots from fungal attack by recruiting an army of protective bacteria into their surrounding soil. Although they don’t as yet know exactly how it’s done, these scientists think that when confronted by root pathogens, plants actively enlist the aid of protective microbes to reinforce their innate defenses.

Microbial teamwork protects sugar beets from R.solani

When R. solani-suppressive soil around the roots, or rhizosphere, of sugar beets was analyzed, Raaijmakers and colleagues found it rich in γ-Proteobacteria, specifically members of the Pseudomonadaceae. But Pseudomonadaceae are not working alone, he emphasizes “several other kinds of bacteria including the Burkholderiaceae, Xanthomonadales, Actinobacteria and Firmicutes all harbor members with effective anti-fungal weaponry and act together with Pseudomonads as protective bacterial consortia.” Moreover, bacteria lacking antifungal-producing ability in isolation can become synergistic when part of a consortium, says Rodrigo Mendes from the Brazilian Agricultural Research Corporation in Jaguarina, the study’s lead investigator. “This is why,” he adds, “it’s so difficult to pinpoint which bacteria are doing what in when it comes to disease suppressive soils.”

Plants, bacteria and archaea work together as a single organism

Like other eukaryotes, plants and their highly-structured microbial consortia or “microbiomes” behave as ‘super-organisms. “Plants rely on soil microbes for specific functions and traits,” say the researchers. “In return the plants exude up to 21% of their photosynthetically-fixed carbon into the root soil interface, or rhizosphere, which feeds the microbial symbionts and influences their activity and diversity.” What was extremely surprising, according to Mendes, “was the unexpected similarities between healthy plant and healthy human gut microbiomes. Not only do the same ‘good’ bacteria act protectively in both systems but there is a similar abundance ratio among the participating bacteria groups,” he adds.

Microbial fingerprinting helps identify >33,000 microbial members in R. solani--resistant soil

PhyloChip: Image courtesy Second Genome

By linking culture-independent and culture-dependant analyses these scientists identified more than 33,000 bacterial and archaeal members in the highly-structured protective sugar beet microbiome — “a richness surpassing any described in previous studies,” say Raaijmakers and Gary L. Andersen at the Berkeley National Laboratory (LBNL) in California who participated in this investigation. The culture-independent testing was done with a new high-density microarray that Andersen developed called the “PhyloChip” which recognizes  the 16S ribosomal RNA gene present in all bacterial and archaea DNA but varies enough from microbe to microbe to “fingerprint” individual cells.

Zooming in on Pseudomonads

Culture-based research had already identified Pseudomonadaceae as natural suppressors of two important fungal predators, Fusarium oxysporum and Gaeumannomyces graminis, and PhyloChip analysis pointed to a prominent role for these bacteria as a suppressor of R. solani as well. Thus the researchers focused their attention on these bacteria, aiming to identify how they make their protective chemical weapon.

First they proved that disease suppression was microbial by sterilizing field samples of R. solani-suppressive soil with heat or gamma radiation and mixing it with resistant soils to show that the resistance could be transferred.  In the year before, sugar beets grown in the suppressive soil were sickened by R. solani infection indicating that, as with other resistant soils, a disease outbreak is needed for soil to acquire protective microbes.  Then Pseudomonadaceae were randomly selected from the naturally occurring R. solani-suppressive soil and a particularly active antifungal strain, SH-C52, was isolated. The scientists created mutants with no in vitro anti-fungal activity from this strain, enabling the researchers to trace wild-type activity to a nonribosomal peptide synthetase (NRPS) gene encoding synthesis of an antifungal chlorinated lipopeptide. One important use of this information would be the design of a “natural” anti-fungal agent which could be used to protect a variety of crops from R. solani infection.

Another more immediate practical application of this study’s results is the employ of specific Pseudomonadaceae and their microbial collaborators as ‘probiotics’ in farming. “This is already being done to some extent,” says Raaijmakers, “but our recent findings give us a much greater diversity of bacteria to choose from than we ever imagined.”

It is important to Steven E. Lindow at the University of California at Berkeley that this work demonstrates how modern microbial methods apply to the study of plant disease control. “It also confirms the power of the PhyloChip,” he adds, noting that its use “verifies the important roles of Pseudomonads in disease suppression. They were fingered years earlier using culture-based tests which we now know have major limitations,” says Lindow, explaining that a great deal of work since has been based on their assumed importance and this has now proved valid.

How microarray-based chip technology works

Chips enable researcher to do metagenomics -genomics on a huge scale- without the need to grow the microbes in a laboratory. (This is important because the vast majority of wild cells do not thrive in captivity.) The method relies on detection of the 16S ribosomal RNA (16S rRNA) gene, a technology pioneered by Norm Pace who is now at the University of Colorado. This important housekeeping gene is present in most if not all bacteria but varies enough to ‘fingerprint’ specific cells and track microbial communities over time and space.

Microbial DNA (or, rarely, RNA) is first extracted from samples such as soil, water and stool. Millions of copies of the 16S ribosomal RNA genes in the samples are amplified with PCR (Polymerase Chain Reaction), placed inside a chip and incubated. The PhyloChip array used in the study described above has a million clusters of short DNA strands each with a different sequence corresponding to the variations found in the 16S rRNA sequences. The results are then matched to the 16S RNAs in a large publicly available database and the corresponding microbes identified.

Briefly:  Nucleic acids are extracted from a sample → 16S genes are amplified with PCR → the PCR product is fragmented → the DNA (or RNA) is labeled and incubated inside a PhyloChip and then washed → the chip is laser scanned to determine where the DNA or RNA “stuck,” according to Todd DeSantis at LBNL.

Note:  Pseudomonads are very talented, they swim, crawl, and walk around on little pili to explore their surrounds, click here to see a daughter cell get up and walk away from mom.

Research details about sugar beets and the bacteria that protect them can be found in:

Mendes  R,  Kruijt M, deBruijn I, Dekkers E, et al. Deciphering the Rhizosphere Microbiome for Disease-Suppressive Bacteria. In Science (27 May 2011); 332, pp.1097-1100.

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