Impact of fresh root material and mature crop residues of oilseed rape (Brassica napus) on microbial communities associated with sub-

sequent oilseed rape

Israr khan

Soil science lab

Gyeongsang National University

South Korea

Introduction

Short-rotation cropping

Short comings of the short-rotation cropping

• Also called as monoculture• Prevalent in conventional arable farming

Importance

• Technological advances• Government incentives,• Economic market trends• Retailer and consumer demands

• Exhibit yield decline compared with those grown in longer, more diverse rota-tions, or grown for the first time

Soil nutrients, pathogens or pests are not always responsible for the loss of pro-ductivity

Introduction

Oil seed Rape (OSR) Brassica napus• A bright-yellow flowering member of the family Brassicaceae• Consumed in China as a vegetable

Cultivation and Uses

• Grown for the production of animal feed, vegetable oil for human consumption, and biodiesel

• Used as a break crop for wheat • Leading producers include the European Union, Canada, the United States, Australia,

China, and India

• Rapeseed was the third-leading source of vegetable oil in the world in 2000, after soybean and palm oil

• The world's second-leading source of protein meal• Form one-fifth of the production of the leading soybean meal

United States Department of Agriculture revelations

Introduction

Reasons for yield decline• Not always clear • Soil and rhizosphere microorganisms are often implicated• Build-up of soil-borne pathogens or a shift towards a more deleterious microbial

community with repeated cropping

Root exudates & Rhizodeposits • Play an important role in recruiting specific microorganisms within the rhizosphere

from the surrounding soil• Root exudates are plant species specific• Leads to the development of particular rhizosphere microbial communities associ-

ated with different plant species, including arable crops

Role of rhizosphere microorganisms in Agriculture• Interaction of microorganisms with the host plant and other microbial community

members• Important due to the potential impact on plant growth, health and crop yield

Introduction

Decoupling root-microbime associations• By using fungicide• Improve rhizosphere suppressiveness to pathogens through inoculation with antago-

nists• Manipulation of rhizosphere microbial communities to provide benefit agricultural

crops Study of rhizosphere microbial diversity• Impact of short-rotation cropping of OSR on microbial diversity have been studied• Significant shifts in soil and rhizosphere fungal and bacterial community was observed • Different rotational frequencies of OSR grown in a long-term field trial

Microorganisms associated with OSR plant material affect the development of soil and rhizosphere microbial communities associated with a subsequent OSR crop ?

Introduction

Objective of the study

1. To determine the effect of repeated inputs of fresh detached OSR root material on

soil and rhizosphere microbial diversity associated with subsequently planted

OSR

2. To determine the influence of field-derived mature OSR crop residues and their

associated microbial communities, on the development of microbial communities

associated with living roots of subsequently grown OSR

Materials and Methods

1. Effect of fresh detached OSR root material on soil and rhizosphere microbial di-versity

• To investigate the effect of repeated inputs of fresh detached OSR root material on soil and rhizosphere microbial communities in the same soil

• Work was conducted in as glasshouse bioassay to allow for a rapid cycle of successive plantings of OSR

• Microbial communities were grown for 4 weeks each time and were monitored over six repeated vegetative cycles of OSR

1.1 Soil source and preparation

• Field soil was collected from plots that had been cropped to wheat for 3 years in Mor-ley, Norfolk, UK

• Soil type was a sandy clay loam with a pH of 6.6 and available P, K, Mg and SO4 2− of 30.6 mg kg−1 respectively

• Soil was sieved (32.4, 111, 28 and 5 mm) to remove stones before glasshouse exper-iment

• Soil moisture content was determined and adjusted to 15 % initially

Materials and Methods

1.2 Experimental setup and sampling procedure

Three main treatments1. unplanted control (soil only)2. OSR seedlings grown and plant material removed from the soil after each of six

(vegetative) replanting cycles3. OSR seedlings grown and the detached root material retained in the soil after

each of six replanting cycles (roots retained; determining the effect of both fresh detached root material and rhizodeposits).

• Six separate pots (size 70×70×80 mm (deep)) were setup initially, to allow for an individual pot to be sampled at each of the six replanting cycles (total of 18 pots per replicate)

• Four complete replicates were set up (total of 72 pots).

Materials and Methods

• OSR (Brassica napus) was planted in each pot for the roots removed and roots-re-

tained treatments, with six seeds planted per pot.

• Seedlings were grown for 4 weeks in each cycle

• During the first vegetative cycle, the roots-removed and roots-retained treatments

were essentially the same.

Materials and Methods

1.2 Molecular analysis

1. DNA extraction and terminal restriction fragment length polymorphism (TRFLP) analyses

2. DNA was extracted from bulk soil and rhizosphere samples collected throughout the experiment, using the FastDNA® SPIN Kit for Soil (MP Biomedicals)

3. PCR amplification universal to the internal transcribed spacer (ITS) region of fungi or the 16S rRNA genes of bacteria.

4. Purified DNA was digested with restriction enzyme Hha which produced the most peaks with an even distribution during TRFLP analysis

5. TRFLP analysis was carried out on an automated sequencer6. Terminal restriction fragments (TRFs) generated by the sequencer were analyzed

using GeneMarker 1.60, and the percentage of the total peak height was calcu-lated for each peak

7. Dominant TRFs were identified through clone libraries8. identified, TRFs are referred to by organism name

Results

Fig. 1 Shannon diversity indices for (a) fungal and (b) bacterial communities in an oilseed rape-re-planting experiment conducted over six vegetative cycles (each cycle lasting 4 weeks). Bars are standard error of the mean. Microbial communities were analyzed from samples (0.5 g) taken for bulk soil (soil) and for a mixture of root material and rhizosphere soil (rhizosphere)

Results

Fig. 2 MDS plot of TRFLP data indicating shifts in microbial community compositions for a fungi in bulk soil, b fungi in rhizosphere samples, c bacteria in bulk soil and d bacteria in rhizosphere samples. Microbial communities were analyzed from samples (0.5 g) taken for bulk soil (soil) and a mixture of root material and rhizosphere soil (rhizosphere). MDS analyses were derived from a Bray-Curtis similar -ity matrix constructed with percentage peak height data of TRFs

Results

Fig. 3 Average relative abundance of Olpidium brassicae (TRF 284) in fungal communities associated with oilseed rape, over six repeated vegetative cycles in the same soil

Results

Fig. 4 Relative abundance of terminal restriction fragments (TRFs) contributing >5 % to the difference between rhizosphere bacterial communities associated with oilseed rape (OSR)

Results

Fig. 5 Relative abundance of terminal restriction fragments (TRFs) contributing >5 % to thedifference between rhizosphere bacterial communities associated with oilseed rape (OSR), grown in a sequence of vegetative cycles

Results

Fig. 6 MDS plots of TRFLP data 2D Stress: 0.03 indicating differences in a fungal and b community compositions associated with soil, shoot residues and root residues at the start of the experiment;

Results

Seedlings were grown for four weeks at a time, over six vegetative cycles. After each cycle, roots were either re -moved from the soil (effect of rhizodeposits only), or retained in the soil for the subsequent vegetative cycle (ef -fect of rhizodeposits and fresh detached root material). Microbial communities were analysed from samples (0.5g) taken for bulk soil (Soil), and a mixture of root material and rhizosphere soil (Rhizosphere)* denotes significant differences between sequential vegetative cycles (P < 0.05) Numbers were set in italics to indicate significant differences both P values and associated R values

Conclusion

Irrespective of the range of inputs returned to the soil following cultivation of OSR

(including fresh root material, rhizodeposits and a variety of field-derived mature

crop residues), the rhizosphere fungal community of OSR was consistently domi-

nated by O. brassicae when OSR was planted in the same soil.

Its interactions with other soil and rhizosphere microorganisms, as well as the

host crop OSR, to gain a better understanding of the multi-trophic interactions that

affect the performance of this globally important crop when grown in short rota-

tion..

Future Research

Thanks for listening

‘Essentially, all life depends upon the soil….There can be no life without soil and no soil without life; they have evolved together’ . Charles E. Kellogge, USDA Yearbook of Agriculture, 1938