Dr. Daniel K. ManterSoil Management and Sugar Beet Research Unit

USDA, Agricultural Research ServiceFort Collins, CO

Development of a Predictive Gene Index

• Soil health, also referred to as soil quality, is defined as the continued capacity of soil to function as a vital living ecosystem that sustains plants, animals, and humans. This definition speaks to the importance of managing soils so they are sustainable for future generations.

Source: NRCS website

• Physical Indicators

• Chemical Indicators

• Biological Indicators

Physical

Biological

Chemical

All three characteristics are directly linked and may be mediated by microbial activity.

Pathogen

Host Environment

Disease

Pathogen

Host

Disease

Effect of soil properties on plant disease

Biological Physical Chemical

++ + ++

++ - +

++ - -

++ + ++

Plant nutrition

Plant defense(ISR)

Plant defense (SAR)

Pathogen growth(antagonism, etc.)

Enzyme Activity

Respiration

Gene expression

COMPOSITION

Abundance

FUNCTION

Characteristics of the soil microbial community

MBC / MBNPLFADNA

Next-gen sequencing

• Taxa-specific abundance

• Species richness & diversity

• GIBBs: Gene Index of Beneficial Bacteria▪ Target beneficial bacteria/genes in soils.

▪ Simultaneously quantify multiple indicators (genes) using a single assay

▪ Expandable assay that can add new modes of action (genes) as they are identified.

▪ Target molecule▪ 16S gene with function mapping

▪ Metagenome (all genes)

▪ Transcriptome (mRNA)

Typical 16S gene marker survey

DNA

Cellvibrio vulgaris (AF448513)

OTU 30 (9130 42)

OTU 79 (95265 1582)

OTU 3 (9130 828)

OTU 14 (9130 1311)

OTU 5 (9130 1059)

OTU 63 (95265 1013)

Cellvibrio mixtus (AF448515)

Pseudomonas syringae (EU906856)

Pseudomonas fluorescens (AF134705)

OTU 70 (9130 1760)

Pseudoxanthomonas sp. (EU276093)

Pseudoxanthomonas mexicana (AF273082)

OTU 6 (9130 1075)

OTU 38 (9130 1016)

OTU 17 (9130 1023)

Acidovorax sp. (EU304287)

Acidovorax defluvii (DQ358210)

OTU 80 (9130 102)

Devosia riboflavina (AF501346)

Devosia sp. (EF433462)

OTU 13 (9130 1111)

Sphingopyxis witflariensis (AJ416410)

Sphingopyxis panaciterrae (AB245353)

OTU 86 (9130 1505)

Flavobacterium succinicans (AM230493)

OTU 32 (9130 1706)

OTU 47 (9130 909)

Flavobacterium sp. (AM934668)

OTU 73 (9130 1070)

qPCR

Starting DNA concentration =>16S rRNA copies per sample

16S rRNAExtract

Building a gene index Map each taxa to a reference genome (PICRUSt: Langouille et al. 2010).

Identify genes/functions present in the community.

0

1

2

3

4

5

6

Gene 1 Gene 2 Gene 3 Gene 4

Site 1

Site 2

Site 3

• C decomposition• b-glucosidase

• xylan 1,4-b-glucosidase

• cellulase

• endo-1,4-b-xylanase

• cellulose 1,4-b-cellobiosidase

• N decomposition• amidase

• urease

• P decomposition• acid phosphatase

• alkaline phosphatase

• S decomposition• arylsulfatase

A list of beneficial bacterial genes/enzymes

• N fixation• nitrogenase

• P solubility• pyrroloquinoline-quinone synthase

• Root growth• ACC deaminase

• indolepyruvate decarboxylase

• Siderophore

• 2,3,-dihydro-2,3-dihydoxybenzoate dehydrogenase

• Salicylate synthetase

• Isochorismate pyruvate lysase

Western Regional Potato Trials

CSU wheat variety drought trials

Examined the effect of cropping intensity under no-till management for 25 years.

Baseline (start of study)WF: wheat-fallow (1986)

25 years of no-till (2009)WCF: wheat-corn-fallowOPP: continuous summer cropCRP: native grassland mix

A disease suppressivity example

• Examined the effect of summer cover crops(mustard, canola, honeysweet,

sordan 79)

• Location: San Luis Valley, CO• Crop: potato• Planting Date: 2005 & 2006

• Crop yield• Nutrient acquisition• Nematode control• Disease suppression

Yield: 12-30% increase

Quality: 40% tubers were 8 oz. or larger

Income: $60-$400 per acre increase

- Effect attributed to increased nutrient efficiency (Delgado et al. 2007)

E5.4.4.2: isochorismate synthase

E4.2.99.21: salicylate synthetase

E1.3.1.28: 2,3,-dihydro-2,3-dihydoxybenzoate dehydrogenase

Phenylalaninebiosynthesis

E5.4.4.2

chorismate

isochorismate

E4.2.99.21

YersiniabactinPyochelinMycobactin

VibriobactinBacillibactinMyxochelinEnterochelin

Function

• Cation chelation

Response

• Microbial & plant nutrient acquisition

• Disease suppression

E1.3.1.28

salicylate 2,3-dihydroxy-benzoate

All cover crops increased

microbial biomass &

soil siderophore content

Cover crops: Disease suppression

Bacterial isolates were also cultured from each soil and measured for in vitro biocontrol activity

0

5

10

15

20

25

Fallow Honey Canola Mustard Sordan

Iso

late

s (#

)

>80

40-80%

20-40%

10-20%

Growth inhibition

Sordan soil had the highest number of isolateswith biocontrol activity.

Mapped genera of beneficial bacteria

A Disease suppressivity example

Soil slurries (biological) from the previous samples were added to 7 day-old potato plants grown in sterile potting media

4-week plants were inoculated with Rhizoctoniasolani

Measured disease development

1 = no lesions

2 = slight lesion development on the lower part of stem

3 = stem lesions moderately severe

4 = lower stem completely girdled by lesion

5 = plant death

Siderophoresynthesis genes

Index is significantly correlated with direct measurement of siderophore activity

Sordan 79 provided the most benefit

• Yield

• Quality

• Nutrient-use efficiency

Soil biology

• Microbial biomass

• Sidreophore production

Gene index of siderophore bacteria was highly correlated with:

• Soil activity

• Disease suppressivity of soil

Flexible▪ Single assay providing an index/metric of beneficial bacteria genes

▪ Any potential gene of index can be easily included

Source material▪ DNA – greater stability in environment; not sensitive to weather events

▪ RNA – includes assessment of gene expression

• Sensitive to management▪ Tillage / cropping intensity

▪ Cover crops

▪ Crop species / cultivars

• Software TooloDesktop version 1.1 currently

availableoPublic web-server in beta testing

• ARS Soil Biology Database

• CapabilitiesoSequence data processing and

analysisoGIBBs integrationoData storage and retrieval

www.myPhyloDB.org

http://nrrc.ars.usd.gov/myPhyloDB/home/