This study examined the endosphere and rhizosphere microbiomes of wheat plants grown in soils infested with the pathogenic fungus Rhizoctonia solani and challenged with Streptomyces biocontrol agents. Wheat seeds were treated with three Streptomyces strains and planted in soils with varying levels of R. solani infestation. Over 20 weeks, 233 root and soil samples were analyzed. The microbiomes changed over time, with some bacteria and fungi increasing. Biocontrol strains promoted plant growth and modulated the root microbiome by decreasing some bacteria like Paenibacillus and increasing other microbes. Soils with highest R. solani levels had more fungal pathogens and plant disease. Application of beneficial Streptomy

1. Decoding Wheat Endosphere– Rhizosphere Microbiomes in
Rhizoctonia solani – Infested Soils Challenged by Streptomyces
Biocontrol Agents.

2. Abstract
The endosphere and the rhizosphere are pertinent milieus with microbial communities that perturb the
agronomic traits of crop plants through beneficial or detrimental interactions. In this study, they challenged these
communities by adding Streptomyces biocontrol strains to wheat seeds in soils with severe Rhizoctonia solani
infestation. Wheat plants were grown in a glasshouse standardized system, and the bacterial and fungal microbiomes of
233 samples of wheat roots (endosphere) and rhizosphere soils were monitored for 20 weeks from seed to mature plant
stage. The results showed highly dynamic and diverse microbial communities that changed over time, with
Sphingomonas bacteria, Aspergillus, Dipodascus and Trichoderma fungi increasing over time. Application of biocontrol
Streptomyces strains promoted plant growth and maturation of wheat heads and modulated the root microbiome,
decreasing Paenibacillus and increasing other bacterial and fungal OTUs. The soils with the highest levels of R. solani
had increased reads of Thanatephorus (Rhizoctonia anamorph) and increased root disease levels and increased
Balneimonas, Massilia, Pseudomonas and unclassified Micrococcaceae. As enter the era of biologically sustainable
agriculture, it may be possible to reduce and limit the effects of serious fungal infestations by promoting a beneficial
microbiome through the application of biocontrol agents during different periods of plant development.

3. Introduction
N.E. Borlaug
• Wheat is the first and foremost staple food crop in the world
• It contributes 40% of cereal food production
• World Production of wheat is 774 Million Tonnes (MT)
(FAOSTAT, 2018)
• Development of healthy societies and has supported
economic and social stability

4. Wheat root rot- Rhizoctonia solani
(Thanatephorus cucmeris)
Disintegration of root
Spindle lesion formed
at collar region
Sclerotia
Bare patches

5. Systematic Classification
Domain – Eukarya
Kingdom – Fungi
Phylum – Basidiomycota
Class- Agaricomycetes
Order - Cantharellales
Family – Ceratobasidiaceae
Genus – Rhizoctonia
Species - solani (Krik et al., 2011)

6. Rhizosphere
Region of soil around the
root surface (1-5mm)
Place where contains
maximum microbial diversity compares
non rhizosphere soils
Rhizoplane
Rhizoplane
microorganisms are found on the
surface of plant roots.
‘R-E Sphere’

8. Use of living organism(macro/micro) to
suppress the population of harmful or
pathogenic organisms.
Biocontrol

9. Techniques used in this study
Illumina Sequencing
High throughput DNA SequencingTechnique.
Employs Micro and Nanotechnologies
Reduce sample size low Reagent cost less
Massive parallel sequencing
Sequence the thousands of sequences at once.
Produce enormous amount of data.

10. Flow cell
Flow cell is a base cell which made up of several lawn and it contained millions and millions
of adaptor (short oligonucleotide around 10-15 nucleotides long)
Function – Sheared DNA which binds to the surface and it leads to upcoming procedure of
sequencing
Flow cell
Lawn
Adaptor

11. Steps used – Illumina Sequencing
Sample Preparation
Cluster Generation
Sequencing
Data analysis

12. Illumina
Seq.
Diagram
Image retrieved from http://res.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf

13. Contd…

14. Materials and Methods
Bulk soil was collected from top 10cm of 100m2 at Waikerie, South Australia (34°14’32.91”S, 140°
5’44.31”E) (Majorly Rhizoctonia sp. infested soil)
Soil was air dried and sieved to < 2mm prior to use of pot bioassay
Pots were prepared based on testing time - 600g for 4 weeks, 1000g for 8 weeks, 1125g for 12
weeks, 2000g for 16 weeks, and 4800g for 20 weeks.
Pot Bioassays in Glasshouse

15. Wheat seeds were treated with Streptomyces sp. strains as F5, F11 and EN16 by using xanthan
gum.
Then, sowing was taken the pot and carefully separate the pot based on testing time (4th,8th ,12th,
16th and 20th week)
BCA treatments and soil infestation levels were run with four independent replicates arranged in a
randomized complete block design.
Half of bioassay pots were artificially made Rhizoctonia sp. infestation.

16. At a time of harvesting, also record the disease incidence of root rot on seminal and nodal region of
roots using a 0–5 disease scale (0 = healthy roots,5 = highly diseased and damaged roots.
At 4, 8, 12, 16, and 20 weeks, plants were carefully harvest, samples were collected from
rhizosphere soil, either seminal or nodal roots for microbiome bioassay

17. Design of Experiment
Randomized Complete Block Design
4th Week N.S. + BCA1 N.S. + BCA3 N.S. + BCA2 N.S. + Control
R. solani infested
soil + BCA 3
R. solani infested
soil + BCA 2
R. solani infested
soil + BCA 1
R. solani infested
soil + control seed
8th Week N.S. + BCA2 N.S. + BCA 1 N.S. + BCA 3 N.S. + Control
R. solani infested
soil + BCA 1
R. solani infested
soil + BCA 3
R. solani infested
soil + BCA 2
R. solani infested
soil + control seed
12th Week N.S. + BCA3 N.S. + BCA 2 N.S. + BCA 1 N.S. + Control
R. solani infested
soil + BCA 1
R. solani infested
soil + BCA 2
R. solani infested
soil + BCA 3
R. solani infested
soil + control seed
16th Week N.S. + BCA3 N.S. + BCA 1 N.S. + BCA 2 N.S. + Control
R. solani infested
soil + BCA 3
R. solani infested
soil + BCA 2
R. solani infested
soil + BCA 1
R. solani infested
soil + control seed
20th Week N.S. + BCA3 N.S. + BCA 1 N.S. + BCA 2 N.S. + Control
R. solani infested
soil + BCA 1
R. solani infested
soil + BCA 3
R. solani infested
soil + BCA 2
R. solani infested
soil + control seed
Abbreivation
N.S. = Native soil (Waikerie, South
Austra.)
BCA 1 = Streptomyces sp. strain F5
BCA 2 = Streptomyces sp. strain F11
BCA 3 = Streptomyces sp. strain EN16

18. DNA Extraction and sequencing
Collection of 16 random samples of root, rhizosphere and seeds of wheat plant
Each sample were subjected to CTAB DNA Extraction. Obtained final DNA was
suspended in TE buffer
Polymerase chain reaction (PCR) was performed by following standards = 35 cycles
of 95°C @ 30 s; 58°C @30 s; and 72°C @60s by using KAPA HiFi PCR
Thermocycler

19. Primers prepared from
a)from bacterial community targeted the V3–V4 regions of the 16S rRNA genes with 341F and
806R primers
b)the fungal community that targeted the ITS1 region with ITS1F and ITS2F primers
After PCR, Samples were pooled and proceed the next step (i.e.) Sequencing by using Illumina
MiSeq System and analysis the variance.
Sequences are categorized and arranged into defined the OTUs of reference species.

20. Thermo cycler

21. Results
i) The Microbiomes Endosphere and Rhizosphere of wheat crops change over time in a
predictable manner, even in soils with severe Rhizoctonia infestation
• Total 1213983 bacteria and 793412 fungal sequences were identified
• Those sequence were organized into 6880 bacterial and 861 fungal OTU’s
• These sequences consisted of 628 bacterial and 204 fungal taxa (assigned at the genus)
• A set of 16 genera of bacteria and 7 fungi were found in all samples. (i.e.)
Agrobacterium, Pseudomonas, Streptomyces, Fusarium etc.

22. Relative abundance
of microbes in all
samples

23. Agglomerative (Bottom up approach) cluster shows maximum diversity
present in rhizosphere soil, followed by roots and then seeds
Rhizosphere soil > Roots > Seeds

24. Shannon diversity index and Margalef richness (at genus or higher taxonomic level) for wheat root and rhizosphere soil
samples; average (minimum and maximum values).
Shannon diversity Shannon diversity Margalef richness
Margalef
richness
Root Rhizosphere Root Rhizosphere
Sampling
time (weeks)
All 4.25 (2.32–6.02) 5.52 (4.68–6.04) 86 223
Seed stage 3.41 (2.64–4.31)* 5.79 (5.08–6.25)* 31* 345*
4 3.29 (2.32–4.14) 5.39 (4.72–5.66) 26 188
8 4.24 (3.49–4.71) 5.49 (5.28–5.77) 76 221
12 4.73 (4.01–5.43) 5.51 (4.68–5.75) 132 230
16 4.50 (3.21–6.02) 5.67 (5.44–6.04) 93 252
20 4.82 (3.74–5.53) 5.51 (5.12–5.88) 140 219
Biocontrol
treatment
Control 4.26 (3.02–5.53) 5.54 (4.72–6.04) 82 227
F11 4.24 (2.32–5.37) 5.51 (4.68–5.76) 86 223
EN16 4.36 (2.66–6.02) 5.51 (5.17–5.88) 93 215
F5 4.15 (3.15–5.16) 5.53 (5.12–6.00) 83 228
Rhizoctonia
soil level
Low level 4.38 (2.99–5.28) 5.59 (4.68–6.04) 97 223
High level 4.10 (2.32–6.02) 5.54 (5.12–5.76) 73 227

25. In this experiment, strain F5 did not perform well , it
showed as similar like control plants, but strains EN16
and F11 gave positive results
• higher biomass at later stages,
• earlier formation of wheat heads and
• lower root disease indexes (more evident with EN16-
coated seeds)
20 weeks
Impact of biocontrol Streptomyces strains on Rhizoctonia sp. and Other OTU’s.
16 weeks

26. Disease incidence index
Grade Description
0 No infection observed
1 Lesion limited to lower 20% of the height of the plant
2 Lesion limited to 21-30% of the height of the plant
3 Lesion limited to 31-45% of the height of the plant
4 Lesion limited to 46-65% of the height of the plant
5 Lesion limited to more than 65% of the height of the
plant

27. Table S1
Rhizoctonia-disease
severity
Waikerie soil + control seed 2.4 ± 0.79
Waikerie soil + seed with F11 2.7 ± 0.75
Waikerie soil + seed with EN16 1.9 ± 0.62
Waikerie soil + seed with F5 2.4 ± 0.52
Waikerie R. solani infested soil + control seed 4.4 ± 0.48
Waikerie R. solani infested soil + seed with F11 4.7 ± 0.11
Waikerie R. solani infested soil + seed with EN16 3.9 ± 0.10
Waikerie R. solani infested soil +seed with F5 4.7 ± 0.83
Tables S1, S2 and S3 show the values of Rhizoctonia disease severity at 4 weeks, weight per plant at 16
weeks and wheat heads per pot at 20 weeks, respectively.

28. Table S2 Weight per plant (mg) at
16 weeks
Waikerie soil + control seed 483 ± 51.0
Waikerie soil + seed with F11 602 ± 158.9
Waikerie soil + seed with EN16 615 ± 51.4
Waikerie soil + seed with F5 515 ± 138.7
Waikerie R. solani infested soil + control seed 571 ± 146.9
Waikerie R. solani infested soil + seed with F11 560 ± 150.3
Waikerie R. solani infested soil + seed with EN16 590 ± 123.6
Waikerie R. solani infested soil +seed with F5 558 ± 34.4
Biomass calculation

29. Table S3 Wheat heads at 20 weeks (average per
pot)
Waikerie soil + control seed 4.5 ± 0.5
Waikerie soil + seed with F11 4.0 ± 0.4
Waikerie soil + seed with EN16 5.3 ± 0.9
Waikerie soil + seed with F5 4.0 ± 0.7
Waikerie R. solani infested soil + control seed 3.3 ± 0.3
Waikerie R. solani infested soil + seed with F11 4.3 ± 0.5
Waikerie R. solani infested soil + seed with EN16 4.75 ± 0.9
Waikerie R. solani infested soil +seed with F5 3.5 ± 0.9
Wheat heads per pot

30. EN16-roots in Waikerie
natural soil (8 weeks)
Control roots in Waikerie
natural soil (8 weeks)

31. • Root rot percent
disease incidence
maximum at the 4th
week
• It decreased in the
subsequent weeks
because of increase of
strains of streptomyces
sp.

32. Taxonomic similarity (%) comparing control versus biocontrol-treated roots; taxonomic groups observed in
roots obtained from low and high Rhizoctonia-level soils were also compared.
Control vs.
F11
Control vs.
EN16
Control vs.
F5
Low vs. high
Rhizoctonia
4 weeks 63 64 48 43
8 weeks 88 88 88 88
12 weeks 59 54 69 53
16 weeks 71 44 69 51
20 weeks 67 46 60 36
Bray Curtis
Similarities
and
ANOSIM
analysis

33. The addition of Streptomyces strains F11 and EN16 affected mostly Paenibacillus populations,
reducing their relative abundance drastically over the 4th weeks.

34. Conclusion
• Microbiomes from rhizosphere and endosphere were constantly change over the time.
• Streptomyces sp. strains (EN16 and F11 ) was effective biocontrol of Rhizoctonia solani
• Streptomyces sp. have a ability to cause a shift over microbiomes population. For eg. Streptomyces
sp. affected the microbiome of Paenibacillus sp.

35. Future Prospectus
• Ability of Streptomyces strain to modify rhizosphere and endosphere microbiome can be harnessed for
improving microbiomes in other staple crops as well.
• Apart from its biocontrol activity, the strain can be checked for its plant growth promoting traits and used
as biofertilizer.
• Optimization of its performance under field conditions need to be validated.

36. Thank You

37. References
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(2015). Roots shaping their microbiome: global hotspots for microbial activity.
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