Báo cáo khoa học nông nghiệp " Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for greater farm profitability and environmental benefits " MS6 potx

1

Ministry of Agriculture & Rural Development

Collaboration for Agriculture & Rural Development


013/06VIE

Replacing fertiliser N with rhizobial
inoculants for legumes in Vietnam for
greater farm profitability and
environmental benefits



MS6: High Quality Inoculants Technical Report







September 2009

1
Table of Contents

1. Institute Information 2

2. Contact Officers 2
3. Project Abstract 2
4. Executive Summary 3
5. Technical Report 5
5.1. Introduction of two Australian strains into Vietnam 6
5.2. Protocols for inoculant production, QA and use 16
5.3. Results and evaluation of on-farm demonstration trials 32

2
1. Institute Information
Project Name
Replacing fertiliser N with rhizobial inoculants for
legumes in Vietnam for greater farm profitability
and environmental benefits
Vietnamese Institution
Oil Plants Institute (OPI)
Vietnamese Project Team Leader
Ms Tran Yen Thao
Australian Organisation
NSW Industry & Investment
University of New England
University of Sydney
Australian Personnel
Dr David Herridge
Dr Roz Deaker
Ms Elizabeth Hartley, Mr Greg Gemell
Date commenced
March 2007
Completion date (original)
March 2009

Completion date (revised)
November 2009
Reporting period
December 2008 – September 2009

2. Contact Officer(s)
In Australia: Team Leader
Name:
Dr David Herridge
Telephone:
02 67631143
Position: Professor, Soil Productivity Fax: 02 67631222
Organisation
University of New England -
PIIC
Email:


In Australia: Administrative contact
Name:
Mr Graham Denney
Telephone:
02 63913219
Position:
Manager External Funding
Fax:
02 63913327
Organisation
Industry & Investment NSW
Email:

In Vietnam
Name:
Ms Tran Yen Thao
Telephone:
08 9143024 –
8297336
Position:
Researcher
Fax:
08 8243528
Organisation
Oil Plants Institute (OPI)
Email:











3
3. Project Abstract

4. Executive Summary
Impact of two Australian strains in Vietnam on legume production and
productivity and comparative analysis between local and introduced strains
Part of this project was to evaluate elite international strains across the country and to
compare them with national strains. Included were local and imported strains from
Vietnamese institutes, from NifTAL (USA), ALIRU (Australia), DOA (Thailand), Korea and
Argentina. Several of these strains are currently used in commercial inoculants in Australia
such as CB1809 (soybean) and NC92 (groundnut). We conducted two experimental sets; the
first was in a potted field soil and the second in field trials.
In the potted field soil trial, there were 13 treatments for groundnut (11 groundnut strains, a

+N control without inoculation and –N uninoculated control) and 18 treatments for soybean
(17 soybean strains, a +N control without inoculation and –N uninoculated control). All
strains increased groundnut and soybean nodulation and yield compared to the control
treatments. There were close correlations between nodule number, nodule weight and plant
biomass while correlations between nodulation and plant height were poor. The best strains
were NC92 (Australian commercial strain), GL1 and GL2 (local strains) for groundnut and
CB1809 (Australian commercial strain), SL2, SL1, CJ2 and U110 (old US commercial
strain) for soybean.

The total number of field experiments during 2007–09 was 36 in the 10 provinces. The
experiments were conducted in the main legume-growing areas in Vietnam, from the
highlands in the North, to the Central Coast area to the highlands in the South and Mekong
Delta. The provinces involved were Son La, Nghe An, Binh Dinh, Binh Thuan, DakLak,
Farmers in Vietnam currently fertilise legumes such as soybean and groundnut with N,
rather than inoculate with rhizobia. Replacing fertiliser N with rhizobial inoculants
would save Vietnamese farmers A$50-60 million annually in input costs and, at the same
time, help facilitate the desired expansion in legume production. There would also be
positive environmental outcomes. This project aims to increase production of high-
quality legume inoculants in Vietnam through enhanced production capacity,
implementation of a national quality assurance (QA) program and increased inoculant
R&D. Participating in the project in Vietnam are the Oil Plants Institute (OPI), the
Institute of Agricultural Science (IAS) and the National Institute for Soils and Fertilisers
(NISF), now known as the Soils & Fertilisers Institute (SFI). Institutions in Australia are
NSW Department of Primary Industries and the University of Sydney. Legume inoculant
use by farmers in Vietnam will be increased through the development and
implementation of an effective extension and training program for researchers, MARD
extension officers and farmers. The benefits of inoculants and legume nitrogen fixation
will be demonstrated in the field and communicated through workshops, meetings and
publications. To ensure sustainability of inoculant production and use, the project will
engage the private sector in marketing and ‘pilot production’ of legume inoculants, with

the aim that they may scale-up production and progressively take over supply as the
technology and markets are developed.

4
DakNong, Tay Ninh, Dong Thap, An Giang and Tra Vinh. There were at least 5 treatments in
each experiment:

1. Farmer
’
s practice without N fertiliser
2. Farmer’s practice with N fertiliser
3. Inoculation with Australian strains CB1809 (soybean) or NC92 (groundnut), -N
fertiliser
4. Inoculation with local strain: SL1 (for soybean) or GL1 (groundnut), -N fertiliser
5. Inoculation with local strain: SL2 (for soybean) or GL2 (groundnut), -N fertiliser

The Australian strains were the most effective in terms of nodulation, biomass yield and
grain yield. Compared with the uninoculated control, CB1809 and NC92 increased
nodulation of soybean and groundnut, respectively, by an average of 58%, biomass yield by
30% and grain yield by 29%. Compared to the local Vietnamese strains, CB1809 and NC92,
increased soybean and groundnut nodulation by an overall average of 22%. Biomass yields
were increased by an average of 10% and grains yields increased by an average of 13%.

Protocols for production of high quality inoculants including QA, packaging,
storage, distribution and on-farm application of inoculants
During the two years of the project, technology for inoculant production at the three institutes
(SFI, OPI and IAS) was developed. The principal aim was production of high quality of
inoculants containing >5 x 10
8
rhizobia/g and a maximum 1 x 10

8
contaminants/g. Some
details of the technologies are different between the collaborating institutes depending on
facilities and expertise. To some extent, the inoculant technologies have been adapted from
those used in countries with existing successful inoculant industries, e.g. Australia, US.
The project team has decided that CB1809 and NC92 will be used for inoculant production in
Vietnam as multi-field trials throughout the country showed these strains are the best for
soybean and groundnut. They increased nodule weight, crop biomass and grain yield
compared to local strains tested. In the future, more strain evaluation will likely be done to
try to develop even more effective inoculant strains. It is also proposed that cultures of these
strains will be supplied annually from the independent QA laboratory to private and public
sector laboratories producing inoculants together with protocols for strain maintenance and
production of broth cultures. Details are provided in Section 5.2.

It is likely that peat will be the major inoculant carrier for Vietnam. Details are provided
(Section 5.2) on different peats in Vietnam, their efficacy as an inoculant carrier and the
usefulness of various additives in improving efficacy. Guidelines are also provided on
optimum pH and water content.

An experiment was done to compare different methods of sterilization of peat. After peat
samples were sterilized, some samples were used for direct determinations of contamination.
Other peat samples were cultured in Glucose Peptone broth and then the broths were assessed
for contamination after 3 hrs, 24 hrs, 36 hrs and 72 hrs. Samples were also assessed for
rhizobial numbers. The best treatments in terms of highest numbers of rhizobia and lowest
numbers of contaminants were the autoclaved treatments with autoclaving for 60 min and the
irradiated treatments with 30 kGy the best overall.


5
There are currently no specific standards for rhizobial inoculants in Vietnam, rather there are

standards for nitrogen fixing microbial fertilizers. However, it is very important to have
effective QA of legume (rhizobial) inoculants. A number of modifications to the Vietnam
National Standard for Nitrogen-Fixing Microbial Fertilizers (TCVN 6166-1996) were
justified to make it more relevant to rhizobial inoculants, based on production technology and
efficacy requirements. The new standards largely utilize the well-constructed and
comprehensive framework of the current standard. The proposed name of the standard is the
Vietnam National Standard for Legume Inoculants and contains details on the technical
requirements of the inoculants including labelling as well as methods of testing and
reporting.

Results of demonstration trials and effectiveness of demonstration trials in
improving farmer’s awareness of benefits
A total of 168 demonstration trials have now been conducted in 10 provinces. The
demonstration fields had two treatments: +inoculation with nil or very low amounts of
fertiliser N and –inoculation with farmer’s rate of fertiliser N. Results are summarised in
Appendix 3.

Generally, inoculation of soybean and groundnut increased the profit for farmers, on average
by 4.500.000VNĐ/ha. The size of the benefit varied across the different sites. The increase
was around 500.000VNĐ/ha at the demonstration field of groundnut at Bau Don, Tay Ninh
province, and as high as 14.200.000VNĐ/ha at Chau Thanh, Tra Vinh province. Similarly for
soybean, the profit from inoculation was as much as 11.640.000VNĐ at Duong Minh Chau,
Tay Ninh province. In Dong Thap province the benefit from inoculation was on average
4.900.000VNĐ/ha.

In Dong Thap province, at the Phu Huu vllage, Chau Thanh district where demonstrations
were conducted on a large area of land (61.5 ha) with the participation of 120 local farmers,
yields of soybean increased on average 12.5%, equal to 300 kg seed/ha. Farmers produced
higher incomes around 4.900.000 VNĐ/ha compared to their normal cultivation with N
fertilisers.

The demonstration trials and associated extension/training activities were very effective in
increasing farmer’s awareness of the benefits of inoculants for legume production. Farmers
were invited to the demonstration sites at least once and, in many fields they came for nodule
and biomass samplings as well as at grain harvest time. Overall, there were a total of 3400+
person visits. They were provided extension materials. Also, researchers and extension
officers explained how rhizobia work and the conditions for successful inoculation. Farmers
were very interested in learning about legume nitrogen fixation.

5. Technical Report
The technical report includes comprehensive details of experiments on rhizobial strains,
protocols for production, QA, distribution and application of inoculants and results and
evaluation of demonstration trails, according to the required report headings:

6
• Introduction of two Australian strains into Vietnam, their impact on legume
production and productivity and comparative analysis between local and introduced
strains.
• Protocols for production of high quality inoculants, packaging, storage, distribution
and on-farm application of inoculants and for quality assurance of production.
• Results of field demonstration trials, including assessement of physical and financial
performance, indication of on-going benefits in cropping rotations and the
effectiveness of demonstration trials in improving farmer’s awareness of benefits.

5.1. Introduction of two Australian strains into Vietnam, their impact on legume
production and productivity and comparative analysis between local and
introduced strains

Introduction

Research on legume inoculants in Vietnam has been done since the 1980
’
s at the Hanoi
University and SFI (VASI) in the North and, in the South, at Can Tho University (CTU), IAS
and OPI (now named IOOP). Generally, the objectives of the research were selection of
strains, small-scale production of inoculants and field trials evaluating efficacy of the
inoculants. Each institute focussed on target regions and particular legume crops, such as
CTU in the Mekong Delta with soybean, IAS in the Southern East Region with groundnut
and OPI in the Central Coast and Highlands with groundnut and soybean. Strains proposed
for inoculant production were not tested throughout the country and outcomes of associated
research on production technologies were not shared between the institutions. Thus, even
with a history of legume inoculant research and production in Vietnam, inoculants are
currently not available in the market and farmers are to a large extent unaware of their
potential benefits. Instead, farmers use expensive N fertilisers on their legume crops. Part of
this project was to evaluate elite international strains across the country and to compare them
with national strains. Included were local and imported strains from Vietnamese institutes,
from NifTAL (USA), ALIRU (Australia), DOA (Thailand), Korea and Argentina. Several of
these strains are currently used in commercial inoculants in Australia such as CB1809
(soybean) and NC92 (groundnut). We conducted two experimental sets; the first was in a
potted field soil and the second in field trials.

Methodology

Screening rhizobial strains in pots

The experimental design was a randomized complete block design with three blocks. There
were 13 treatments for groundnut (11 groundnut strains, a +N control without inoculation and
–N uninoculated control) and 18 treatments for soybean (17 soybean strains, a +N control
without inoculation and –N uninoculated control). Information of strains is given in the Table
1. Each strain was grown up in yeast mannitol broth (YMB) for 5–7 days to reach maximum

turbidity (approx. 1 x 10
9
cells/ml). The broths were then injected into sterilized peat and
allowed to stabilise for 1 week. Seeds were inoculated with the peats at the rate of 10
5
–10
6
cells/seed just before sowing.


7
The sandy, infertile soil used in the pots was from Trang Bang district, Tay Ninh province.
The soil was obtained from a depth of 10–15 cm and transported to Binh Thanh experimental
station of OPI. The soil was mixed thoroughly, then sieved using a 5-mm mesh screen. The
soil was then mixed with coir dust (1:1) and lime and allowed to equilibrate for 7 days. Each
pot contained 1.7 kg of the soil mixture.

The soil moisture content at field capacity was determined, then each pot adjusted to field
capacity by adding water. Application of fertiliser was as follows: KH
2
PO
4
- 195 mg/pot;
KCl - 168.4 mg/pot; MgSO
4
.7H
2
O - 22.21 mg/pot; ZnSO
4
.7H

2
O - 20.63 mg/pot;
(NH
4
)
6
Mo
7
O
24
.7H
2
O - 0.81 mg/pot.

We planted 5 seeds/pot and removed 2 young plants after 7 days. The plants were harvested
at 30 days for soybean and at 45 days for groundnut. Numbers of nodules, dry weight of
nodules and dry weight of biomass was determined at harvest.

Table 1. Rhizobial strain information

No. Strain name Target crop Source
1 NC92 Groundnut Australia
2 Tal 179 Groundnut NifTAL
3 P088183 Groundnut Thailand
4 P03818 Groundnut Thailand
5 GL1 Groundnut OPI – local strain
6 GL2 Groundnut SFI – local strain
7 GL14 Groundnut OPI – local strain
8 LAC1 Groundnut SFI – local strain
9 P3 Groundnut OPI – local strain

10 P1 Groundnut OPI – local strain
11 CTP Groundnut CTU – local strain
12 CB1809 Soybean Australia
13 U110 Soybean NifTAL
14 SEMIA 5019 Soybean NifTAL
15 S01015 Soybean Thailand
16 S1059 Soybean Thailand
17 Ach Soybean Argentina
18 YCK Soybean Korea
19 SL1 Soybean SFI – local strain
20 DT2 Soybean SFI – local strain
21 SL2 Soybean SFI – local strain
22 DL1 Soybean OPI – local strain
23 DL2 Soybean OPI – local strain
24 CJ1 Soybean OPI – local strain
25 CJ2 Soybean OPI – local strain
26 S6 Soybean OPI – local strain
27 S37 Soybean OPI – local strain





8
Field experiments

The experiments were conducted in 10 main legume-growing areas in Vietnam, from the
highlands in the North, to the Central Coast area to the highlands in the South and Mekong
Delta. The provinces involved were Son La, Nghe An, Binh Dinh, Binh Thuan, DakLak,
DakNong, Tay Ninh, Dong Thap, An Giang and Tra Vinh. There were at least 5 treatments:

6. Farmer
’
s practice without N fertiliser
7. Farmer’s practice with N fertiliser
8. Inoculation with CB1809 (for soybean) or NC92 (groundnut), -N fertiliser
9. Inoculation with local strain: SL1 (for soybean) or GL1 (groundnut), -N fertiliser
10. Inoculation with local strain: SL2 (for soybean) or GL2 (groundnut), -N fertiliser

Source of strains:

SL1: local strain (soybean) from Can Tho University
SL2: local strain (soybean) from SFI (VASI - from the national microbial strain program)
GL1: local strain (groundnut) from OPI
GL2: local strain (groundnut) from SFI (VASI - from the national microbial strain
program)
CB1809: Australian commercial inoculant strain (soybean) from ALIRU
NC92: Australian commercial inoculant strain (groundnut) from ALIRU

Measurements were: dry weight of nodules, biomass and grain yield. Plot size was at least 20
m
2
with 4 replications. A randomized complete block design was used. Depending on
growing areas, sowing date, land preparation, fertiliser inputs, date of sampling were
different. Details can be sent if required.

Peat inoculants were made by the three Vietnamese institutes (OPI, IAS and SFI) and in
some experiments Australian commercial inoculants were use as a positive control treatment.
Inoculant rates were 1–2 kg/ha for Vietnamese inoculants and 0.25 kg/ha for Australian
commercial inoculants. The inoculants were tested for quality by OPI before conducting

experiments. The seed inoculation method was used throughout. Methods of sampling and
processing of nodules, biomass and grain yield can also be sent if required.

Results and Discussion

Screening rhizobial strains in pots

All strains increased groundnut nodulation and yield compared to control treatments (Table
2). Based on assessments of plant nodulation, there were 3 groups:
- Highest nodulation: NC92, GL1, GL2
- Average nodulation: P12, GL14, P03818
- Lower nodulation: P08183, CTP, P31, LAC1, Tal179
There were close correlations between nodule number, nodule weight and biomass (r
2
=0.82)
while correlation between nodulation and plant height was not significant (r
2
=0.27). The
results showed that NC92, GL1 and GL2 were the best strains. They produced more nodules
and more biomass than other strains.




9
Table 2. Nodulation and growth of inoculated groundnut with different rhizobial strains
Rhizobial strains
Number of
nodules/plant
Dry weight of

nodules/plant (mg)
Plant height (cm)
Dry biomass
(g/plant)
1. NC92 79 b 93 b 33 abcd 2.7 b
2. P08183 29 gh 35 fg 29 cd 1.7 ghi
3. CTP 44 e 52 d 36 ab 2.2 def
4. GL1 113 a 136 a 35 abc 3.8 a
5. P31 35 fg 42 ef 31 bcd 1.7 hij
6. GL2 77 b 91 b 35 ab 2.5 bc
7. P12 54 cd 66 c 35 ab 1.9 fgh
8. GL14 60 c 72 c 32 abcd 1.9 efg
9. LAC1 39 ef 47 de 38 a 1.6 ij
10. Tal 179 27 h 33 g 35 ab 1.5 jk
11. P03818 53 d 63 c 32 abcd 2.4 cd
Control 1 11 i 12 h 27 d 1.2 k
Control 2 5 i 6 h 32 abcd 2.2 de
CV% 10.6 11.4 12.7 8.6
Control 1: uninoculated, without N fertilizer
Control 2: uninoculated, plus N fertilizer (100ppm)
Source: OPI
Table 3. Nodulation and growth of inoculated soybean with different rhizobial strains
Rhizobial strains
Number of
nodules/plant
Dry weight of
nodules/plant (mg)
Plant height (cm)
Dry biomass
(g/plant)

12. U110 47 cd 129 d 38 defg 2.8 d
13. DT2 45 de 121 e 35 defg 2.7 e
14. CB1809 52 bc 149 b 43 abcd 3.3 bc
15. CJ1 35 fg 121 e 41 bcde 2.7 e
16. DL2 30 g 72 h 34 efg 1.6 hi
17. S37 16 h 103 f 50 a 2.3 f
18. SL2 66 a 170 a 42 abcd 3.7 ab
19. CJ2 49 cd 136 cd 42 abcde 2.9 c
20. DL2 35 fg 114 e 37 defg 2.5 e
21. SL1 57 b 141 c 37 defg 3.1 cd
22. YCK 33 fg 77 gh 48 ab 1.7 gh
23. S01015 35 fg 121 e 46 abc 2.6 e
24. SEM 5019 34 fg 118 e 39 cdef 2.6 e
25. DL1 39 ef 83 g 39 cdef 1.8 g
26. S6 21 h 80 g 39 cde 1.7 gh
27. ACH 22 h 69 h 30 g 1.5 i
28. S1059 32 fg 97 f 31 fg 2.2 f
Control 1 9 i 35 i 43 abcd 1.3 j
Control 2 8 i 29 i 37 defg 1.4 i
CV% 13.2 5.1 14.6 4.1
Control 1: uninoculation, without N fertilizer
Control 2: uninoculation, plus N fertilizer (100ppm) - Source: OPI

10
Similarly, inoculated soybean produced more nodules and more biomass compared to
controls (Table 3) with substantial differences amongst the strains. Grouping strains based on
effectiveness of nodulation and yield as follows:
- Highest effectiveness: SL2, CB1809, SL1, CJ2, U110
- Average effectiveness: DT2, CJ1, S37, S01015, SEMÍA 5019, S1059
- Lower effectiveness: YCK, ACH, DL1, DL2, S6

In the first group, biomass amounts were 3 times higher than control treatments. The close
correlation between nodulation and biomass showed that nodules were very effective and
contributed to higher biomass yield (r
2
=0.86) and will likely play an important role in seed
yield increase. The 5 strains CB1809, U110, SL1, SL2 and CJ2 were the best strains for
soybean.

In another experiment conducted in a green house, CB1809 showed high effectiveness in
term of nodulation (Table 4). The strain produced more nodules compared to 3 other local
strains.

Table 4. Nodulation of 4 rhizobial strains for soybean in potted soil

Nodule number/plant No Treament
Total nodule
number
Nodule number
on main roots
Nodule number on
lateral roots
1 Control - - -
2 Inoculation with CB1809 50 25 25
3 Inoculation with SL1 32 13 19
4 Inoculation with SL2 39 13 26
5 Inoculation with SL3 32 11 22
Source: SFI
Field experiments
The total number of field experiments across the project was 36 in the 10 provinces. A
summary of project field experiments during 2007–09 and inoculation effects of CB1809 or

NC92 on nodule, biomass and grain yield is shown in Appendix 1. A small response to
inoculation was determined as less than 20%, from 20 to 40% was a moderate response and
large response was more than 40%.
Graphs 1, 2 and 3 summarise responses to inoculation with strains CB1809 (soybean) or
NC92 (groundnut) for plant nodulation, biomass and grain yield, respectively. Responses
ranged from small to large, depending on field sites.

There were large effects of inoculation on nodulation at 72% of the field sites (Graph 1). At
those sites, nodule weight increased by 43–166%. Nodulation responded moderately at 11%
and, at the rest (17%) of the sites, response were small with an average increase of 11%. The
overall average increase in nodulation using the superior Australian strains was 58%.

11
Graph 2. Range of crop biomass responses to inoculation
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40
Field site
% Response


Graph 1. Range of nodulation responses to inoculation
-20
0

20
40
60
80
100
120
140
160
180
0 5 10 15 20 25 30 35 40
Field site
% Respons
e


With crop biomass, there were large responses (44–86%) to inoculation at 33% of the field
sites, moderate responses (17–40%) at 45% of the sites and small responses (5–19%) at the

12
remaining 22% of sites (Graph 2). Increases in grain yield from inoculation were smaller
than the increases in nodulation and biomass yield (Graph 3). There were large responses
(41–70%) at 20% of sites. Moderate responses (20–40%) were recorded at 62% of the sites
and small responses (4–18%) at the remaining 18% of sites. The overall average increases in
biomass yield and grain yield using the superior Australian strains were 30% and 29%,
respectively.

There were large differences in nodulation, biomass yield and grain yield responses amongst
the rhizobial strains. Australian commercial strains CB1809 (soybean) and NC92
(groundnut) were more effective than local Vietnamese strains at almost field sites
(Appendix 2). Data analysis shows that when the crops were inoculated with CB1809 or

NC92, nodule weight, biomass yield and grain yield increased relative to the local strains at
91%, 94 and 97% field sites respectively. However, the extent of the increase was different
depending on sites and local strains. Graphs 4, 5 and 6 show the increase in nodulation,
biomass and grain yield of soybean and groundnut when inoculated with CB1809 and NC92,
respectively, compared with inoculation using local strains.

The two Australian strains, CB1809 and NC92, increased soybean and groundnut nodulation
by an overall average of 22%, relative to the local Vietnamese strains (Graph 4). Biomass
yields were increased by an average of 10% (Graph 5) and grains yields increased by an
average of 13% (Graph 6), relative to the local strains.

Graph 3. Range of grain yield responses to inoculation
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30 35 40
Field site
% Response



For each of the measures, there were large variations according to the particular site. For
nodulation, the range was 0–70%. For biomass yield, the range was 0–30% and for grain
yield, the range was 0–51%. Yield increased more than 20% relative to the local strains at

18% sites, from 10–20% at 34% sites and from 1–10% at 48% sites.


13
Graph 4. Nodulation increases with CB1809 and NC92 compared to
local strains
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30 35 40
Field sites
% increase
Local strain1
Local strain 2

Graph 5. Crop biomass increases with CB1809 and NC92 compared
to local strains
0
5
10
15
20
25
30

35
0 5 10 15 20 25 30 35 40
Field trials
% Increases
Local strain1
Local strain2


14
Graph 6. Yield increases with CB1809 and NC92 compared to
local strains
0
10
20
30
40
50
60
0 5 10 15 20 25 30 35 40
Field trials
% Increase
Local strain1
Local strain2


5.2. Protocols for production of high quality inoculants, packaging, storage,
distribution and on-farm application of inoculants and for quality assurance of
production
During the two years of the project, technology for inoculant production at the three institutes
(SFI, OPI and IAS) was developed. The principal aim was production of high quality of

inoculants containing >5 x 10
8
rhizobia/g and a maximum 1 x 10
8
contaminants/g.

In the
following we present the current technologies for inoculant production in Vietnam. Some
details of the technologies are different between the collaborating institutes depending on
facilities and expertise. To some extent, the inoculant technologies have been adapted from
those used in countries with existing successful inoculant industries, eg Australia, US.
Strains for production
The project team has decided that CB1809 and NC92 will be used for inoculant production in
Vietnam as multi-field trials throughout the country showed these strains are the best for
soybean and groundnut. They increased nodule weight, crop biomass and grain yield
compared to local strains tested. In the future, more strain evaluation will likely be done to
try to develop even more effective inoculant strains.

Maintenance of strains and preparation of mother cultures

It is proposed for production of inoculants in Vietnam that mother cultures will be provided
annually by an independent quality control laboratory where the strains for production are
maintained in terms of purity, viability and effectiveness (nodulation and nitrogen fixing
ability). After receiving the mother cultures, producers (manufacturers) have to maintain
them for that year of production.


15
The first step after receiving the mother cultures is for the cultures to be transferred into
culture tubes containing YMA (yeast mannitol agar) medium (called sub-mother cultures), at

the same time streaking the cultures onto YMA and CRYMA (congo red yeast mannitol
agar) Petri dishes for purity checking. If the result show that the mother culture is
contaminated then a request to the quality control lab for a new mother culture will be made.
The mother culture should be pure before sending to manufactures but there are risks
associated with transport or handling. Pure sub-mother cultures will then be kept in a
refrigerator by the manufacturers at low temperature (1–4
o
C) until use. They need to be sub-
cultured after 6 months. The number of sub-cultures prepared will depend on the demand of
production. Culturing temperature for the rhizobia is 30
o
C in an incubator. Another method
can be applied when there is a higher demand for production. Transfer the mass culture from
the mother culture to a flask with YM broth, then inoculate at 30
o
C for 5–7 days. Two ml of
the broth culture then are dispensed into 2 ml of 50% glycerol, previously sterilized in
bottles. These sub-mother cultures are stored in a domestic deep-freeze for use before
checking purity by streaking on CRYMA and YMA plates.

Growth of broth cultures

Starter culture: Streak out the culture of the strain to be used onto CRYMA and YMA
Petri plates to check for purity. Place the plates in an incubator at 30°C for 6–8 days. If the
plate streak is pure, aseptically pour about 10 mL of sterile water onto the slope sub-culture.
Close the lid and gently wash the slope with the water. Aseptically, pour the liquid
containing the rhizobia into an appropriately-sized flask containing YM broth. Reseal the
flask with the cotton wool bung and paper bag. Gently mix the broth, and place in an
incubator shaker at 30°C until the broth becomes milky in colour (5-7 days), and the
rhizobial population reaches at least 10

9
/ml broth. The broth in the flask can then be
aseptically poured into a fermentor.

Broth growth media: Medium for rhizobial growth consists of a carbon source, nitrogen
source and minerals. Most rhizobia can utilize pentoses, hexoses, disacharides,
polysaccharides and sugar alcohols even though the carbon utilization properties of rhizobia
vary. Generally, improvements in medium for inoculant production is based on the basic
medium, YMB (yeast mannitol broth). Large scale inoculant production requires cheap and
available ingredients, such as corn steep liquor and proteolyzed pea husks. Table 5 below
shows effects of different growth media on growth of 6 strains of rhizobia. The alternatives
tested by SFI were green bean extract, saccharose and glucose instead of expensive yeast
extract and mannitol. The number of rhizobial cells of the 6 strains yielded higher than
10
9
/ml in the proposed media SX1 and SX2, similar to YMB. The SX1 medium is the best
one in terms of efficacy and economic benefit.

The data are promising and need to be repeated. If rhizobial growth rates on media SX1 and
SX2 are again high, then they can be considered for inoculant production in Vietnam.

Duration of growth: The results showed that the numbers of rhizobial cells increased over
time and reached a maximum at the 7
th
day for all strains (Table 6). Counts were as high as 7
x 10
9
cells/ml.

16
Table 5. Number of cells/ml culture in improved media compared to basic YMB medium

Number of rhizobial cells per ml
Strain
YMB SX1 SX2
CB1809 8,2 x 10
9
1,6 x 10
9
4,2 x 10
9

SL1 4,8 x 10
9
6,8 x 10
9
5,8 x 10
9

SL2 2,7 x 10
9
5,4 x 10
9
1,2 x 10
9

NC92 2,8 x 10
9
4,8 x 10
9
5,6 x 10
9

GL1 5,6 x 10
9
2,8 x 10
9
1,9 x 10
9

GL2 5,6 x 10
9
7,2 x 10
9
5,2 x 10
9


SX1(g/l): green bean extract 50 g; K
2
HPO
4
0.5 g; KH
2
PO
4

0.5 g; (NH
4
)2SO
4
1.0 g; MgSO
4
. 7H
2
O 0.5
g; CaCO
3
1,0g; Glucoza 5,0g; Saccharoza 5,0g; distilled water 1 litre.

SX2: Glucose 10g; KH
2
PO
4
0,5g; MgSO
4
.7H2O 0,25g; CaCO
3
0,5g; Yeast Extract 0,5g; distilled
water 1 litre.

YMB: control – mannitol 10g; yeast extract 1g; 0,5g; KH
2
PO
4
0,5 g; MgSO4. 7H2O 0,5g; NaCl 0,1 g;
distiled water 1 litre

(Source: SFI)

Table 6. Number of rhizobial cells in broth cultures at different growth times

Rhizobial cells/ml
Growth time
(day)
CB1809 SL1 SL2 NC92 GL1 GL2
3 5,2 x 10
7
8,8 x 10
6
1,04 x 10
7
5,2 x 10
7
9,2 x 10
6
1,36 x 10
7

4 8,8 x 10
8
2,48 x 10
7
1,44 x 10
7
2,6 x 10
8
5,8 x 10

7
1,3 x 10
8

5 1,24 x 10
9
4,02 x 10
8
2,4 x 10
8
8,0 x 10
8
2,2 x 10
8
8,4 x 10
8

6 3,2 x 10
9
2,8 x 10
9
1,08 x 10
9
3,6 x 10
9
1,24 x 10
9
1,08 x 10
9

7 7,2 x 10
9
4,8 x 10
9
4,8 x 10
9
4,2 x 10
9
2,6 x 10
9
1,22 x 10
9

8 1,68 x 10
9
1,02 x 10
9
1,28 x 10
9
2,36 x 10
9
1,56 x 10
9
2,4 x 10
9

Source: SFI

pH effects:
Table 7. Effects of pH on the growth of rhizobial strains

Growth of rhizobial strains
pH
CB1809 SL1 SL2 NC92 GL1 GL2
4
- - - - - -
4.5
- - - - - -
5
- - - - - -
5.5
+ ++ ++ ++ ++ ++
6
++ ++ ++ ++ ++ ++
6.5
+++ +++ +++ +++ +++ +++
7
+++ +++ +++ +++ +++ +++
7.5
++ ++ ++ ++ ++ ++
8 - - - - - -
8.5
- - - - - -

17

-: no development
+: weak development (10
4
–10

5
CFU/ml)
++: nomarl development (10
6
–10
7
CFU/ml)
+++: good development (10
8
–10
9
CFU/ml
Source: SFI

Temperature effects:
Table 8. Effects of temperature on growth of rhizobia

Temperature
(ºC)
Growth of rhizobial strains
CB1809 SL1 SL2 NC92 GL1 GL2
25 ++ ++ ++ ++ ++ ++
30 +++ +++ +++ +++ +++ +++
37 + + + + + +
>45 - - - - - -
-: no development
+: weak development (10
4
–10
5

CFU/ml)
++: nomarl development (10
6
–10
7
CFU/ml)
+++: good development (10
8
–10
9
CFU/ml
Source: SFI

Results from Tables 7 and 8 showed pH of medium and temperature effects on the
growth of rhizobia. All 6 strains tested developed well at pH 6.5–7 and at 30
0
C.

Fermentation: Depending on capital investment or available equipment and expertise,
manufacturers will need to consider how big the fermentors should be. Large-scale batch
fermentation has efficiency in terms of time but may be more prone to contamination.
Fermentors can vary in size from 1–2 L (glass flasks on a rotary shaker or with an aerator) to
>1600 L (purpose-built steel fermentors).

Broth cultures can be produced in simple glass fermentors, such as a slightly modified 4-L
Erlenmeyer flask with a sampling port fitted close to its base. A maximum of 2–3 L of
culture medium is added to the flask. To prevent the entry of contaminants, the cotton wool-
packed filters are connected via the air lines. All rubber stoppers and outlet tubes are
autoclaved. The large rubber stopper which holds the air inlet and outlet tubes with their
filters is inserted firmly into the neck of the flask. An aquarium pump is used to supply

aeration. It is connected with the air outlet tube. Air will flow freely through both filters
while bubbling through the broth. The cotton wool in the filters needs to be packed uniformly
but loosely. Over packing the air inlet filter can cause resistance to incoming air and lead to
poor aeration. Over packing the outlet filter can lead to poor air escape and pressure build up
in the fermentor.

The pump is disengaged from the fermentor for sterilisation. Sterilise for 40 minutes at 1 atm
if the broth is 2 L and increase time by 10 minutes for each additional 1 L. After the
fermentor has cooled, remove the clamp from the air inlet tubing and connect the air supply
then check for proper aeration and for leaks in the system. The system is ready for
inoculation with the starter culture. Inoculation is conducted through the latex air inlet with a
sterilized syringe fitted with a needle. The air inlet tubing is surface sterilised with 70%
alcohol or 3% hydrogen peroxide about 2–3 cm above its connection to the glass tube. The

18
needle is inserted downwards into the tubing and the starter culture is injected. The culture
then is incubated at 30
o
C. Use of small fermentors would be appropriate for Vietnam,
particularly when combined with the broth dilution – solid state fermentation. With this, the
broth can be diluted 100 fold. Thus, a 2 L broth can be used to inoculate 10,000 packets (at
rate of 20 mL/packet).

Small steel fermentors are common in industry and are usually sterilized by autoclave. A
fermentor system for culturing rhizobia was designed by NifTAL and uses direct heating by
gas and cooling tubes for time-saving after sterilisation. The body of the NifTAL fermentor
is a pressure vessel with a 141 L total capacity. Working (broth) capacity is 20–100 L.
Details of this fermentor can be accessed through Prof. Nantakorn Boonkerd’s laboratory at
Suranaree University of Technology, Thailand.

Selection of carriers

Carrier plays an important role in solid-based inoculant production. Most inoculants are
produced based on the mixture of the broth culture and a finely milled, neutralized carrier
material. The properties of the good carrier are:

- Supportive to rhizobial growth and survival
- Good moisture absorption capacity
- Easy to process
- Easy to sterilize by autoclave or gramma-irradiation
- Available in adequate amounts
- Inexpensive
- Good adhesion to seeds
- Good pH buffering capacity


Table 9. Characteristics of Sedge Peat used for commercial inoculant production
in the United States

Type Amount Ash analysis (%)
N total (%) 1.62 K 1.12
Organic matter (%) 86.80 P 0.33
Ash (%) 13.20 Ca 5.21
Exchangeble K (ppm) 62.00 Mg 1.14
N - NH4 và NO3 (ppm) 94.00 Fe 2.10
Available P (ppm) 12.00 Si 28.00
pH 4.5 -5.0 Al 6.32
Moisture (%) 7 – 8 Na 0.52
Burton (1979)

Peat is the best researched and most frequently used as carrier materials for inoculant
production. A large number of studies have showed that rhizobia are protected and survive
well in peat. Tables 9 and 10 show physical and chemical analysis of well-researched peats.
These peat were used for commercial inoculant production in U.S.A and Australia. However,
physical and chemical analyses of a peat are only a partial assessement of its suitability as a
carrier. Only a test related to growth and survival of rhizobia can confirm its acceptability.

19
Apart from characteristics at the source such as salinity, clay, organic matter and
contamination with chemical residues, some unknown factors will affect suitability of peat
for use as an inoculant carrier. Peats from different sources should be tested after adjusting to
the same particle size distribution and moisture content (if possible). It is not possible to
judge the suitability of peat from the colour or texture.


Table 10. Characteristics of Bendenoch Peat used for commercial inoculant production in
Australia

Characteristics Range Average
Organic matter (%) 28.8 – 75.4 64.3
Organic C (%) 16.4 – 42.1 36.1
Minerals (%) 10.0 – 16.0 12.1
Soluble salts (%) 0.09 – 1.50 0.87
Cl (%) 0.01 – 0.31 0.11
N (%) 0.89 – 2.30 1.83
K
2
O (%) 0.12 – 0.17 -
P
2

O
5
(%) 0,09 – 0.22 -
Water holding capacity (%) 216 – 522 320
C/N 15.0 – 17.5 16.7
pH 5.8 – 7.8 6.8
Roughley (1970)
Vietnam has many peat mines located throughout the country but has a large range of quality,
from poor (Can Gio), moderate (Binh Phu) to good peat (U Minh) (Table 11).

Table 11. Chacteristics of 3 peat sources in Vietnam

Characteristics Can Gio Binh Phu U Minh
Ash (%) 50 - 70 40 - 60 7 – 9
OM (%) 14 - 21 25 - 40 46 – 51
Acid humic (%) 5 - 8 14 – 18 30
S (%) 1 - 8 0.3 – 1.5 0.25
Source: Peat in Vietnam and use in agriculture (Agriculture Publish House, 1997)

Results in Table 12 show effects of peat sources on inoculant quality. Except Komix 2 the
three other Vietnamese peats produced good growth of rhizobia. The number of rhizobia in
these peat reached ≥ 10
9
cfu/g moist peat, equal to commercial Australian peat. The number
of rhizobia in Komix 2 was only 3.4 x 10
7
cfu/g moist peat at 6 months.

Similarly, Son La and Thai Nguyen peat of the North supported good growth and survival of
rhizobial strains CB1809, NC92, GL2, SL2 (Table 13). The number of rhizobia was ≥ 10

9

cfu/g moist peat.




20
Table 12. Number of rhizobia in different peat sources of the South

Treament Innitial 1 week 2 week 3 week 4 week
1. Australia 59 x 10
7
30 x 10
8
16 x 10
8
20 x 10
8
17 x 10
8
2. Komix 1 40 x 10
7
22 x 10
8
29 x 10
8
32 x 10
8
25 x 10

8
3. Komix 2 18 x 10
7
63 x 10
7
33 x 10
7
42 x 10
7
34 x 10
7
4. DakNong 20 x 10
7
15 x 10
8
19 x 10
8
20 x 10
8
24 x 10
8
5. U Minh 46 x 10
7
25 x 10
8
24 x 10
8
18 x 10
8
22 x 10

8
Treament 2 month 3 month 4 month 5 month 6 month
1. Australia 31 x 10
8
20 x 10
8
14 x 10
8
12 x 10
8
10 x 10
8
2. Komix 1 28 x 10
8
24 x 10
8
15 x 10
8
17 x 10
8
12 x 10
8
3. Komix 2 25 x 10
7
32 x 10
7
10 x 10
7
74 x 10
6

34 x 10
6
4. DakNong 20 x 10
8
15 x 10
8
11 x 10
8
12 x 10
8
78 x 10
7
5. U Minh 25 x 10
8
19 x 10
8
15 x 10
8
14 x 10
8
11 x 10
8
Source: OPI
Soybean strain CB1809

There are a wide range of substitutes to peat, e.g. baggage, filter mud, vermiculite,
polyacrylamide, mineral soils, coal, charcoal, ground plant residues. These materials can be
also incorporated with peat to improve quality. Baggasse, coir dusts and worm casts have
been shown to improve peat quality through increased water holding capacity (Table 14).
The number of rhizobia increased when peat was mixed to worm casts or worm casts plus

coconut coir dusts compared to peat control.
Table 13. Number of rhizobia in two peat sources of the North

Number of rhizobia/g moist peat inocualnts
Rhizobium
strain
Peat source
Initial 1 week 2 week 1 month 2 months 3 months
Sơn La 3,2 x 10
9
1,8 x 10
9
4,2 x 10
9
7,2 x 10
9
8,0 x 10
9
4,0 x 10
9
NC92
Thái Nguyên 2,4 x 10
9
1,3 x 10
9
3,6 x 10
9
7,6 x 10
9
4,0 x 10

9
6,8 x 10
9
Sơn La 1,6 x 10
9
1,2 x 10
9
1,8 x 10
9
2,1 x 10
9
1,4 x 10
9
1,3 x 10
9
GL2
Thái Nguyên 1,2 x 10
9
2,4 x 10
9
1,2 x 10
9
1,1x 10
9
3,6 x 10
9
1,2 x 10
9
Sơn La 6,5 x 10
9

1,8 x 10
9
7,5 x 10
9
3,4x 10
9
3,2 x 10
9
3,6 x 10
9
CB1809
Thái Nguyên 5,3 x 10
9
2,8 x 10
9
5,3 x 10
9
2,2 x 10
9
2,4 x 10
9
1,4 x 10
9
Sơn La 6,6 x 10
9
1,4 x 10
9
1,6 x 10
9
1,5 x 10

9
5,2 x 10
9
1,1 x 10
9
SL2
Thái Nguyên 4,0 x 10
9
1,2 x 10
9
4,8 x 10
9
1,0 x 10
9
1,0 x 10
9
1,2 x 10
9

Source: ISF




21
Table 14. Number of rhizobial cells in different carriers after one month
No Rhizobial strain Carriers
Number of rhizobial cells
(CFU/g)
1 NC 92 Peat 2.7 x 10

7

2 CB 1809 Peat 5.4 x 10
6

3 NC 92 Peat + worm casts 8.3 x 10
8

4 CB 1809 Peat + worm casts 5.4 x 10
8

5 NC 92 Peat + worm casts + coconut coir dust 2.5 x 10
9

6 CB 1809 Peat + worm casts + coconut coir dust 6.8 x 10
8

Source: IAS
1 NC92 Peat
1,2 x 10
8
2 NC92 Peat + molasses + “rare soil”
1,5 x 10
9

3 NC92 Peat + worm casts + coconut coir dust
3,8 x 10
9

Source: ISF

Carrier processing

Particle size of carriers: Peat is currently the preferred carrier for inoculant production in
Vietnam. The project research has shown some additives can be used as mixtures with peat.
They are worm cast and coconut coir dust. Molasses and “rare soil” can be also added as
nutrient additives. The peat is mined, drained if wet peat, screened to remove stones and
roots and then shredded and dried. The peat then is ground in high-speed harmer and passed
through a sifting machine which has a set of sieves: 1mm, 355µm, 150 µm and 75 µm. The
milled carrier will go through the sieves. Particles of 75 µm or thinner make carriers suitable
for seed coating.
pH of carriers: The pH was shown to be critical and acid peats could be amended with
calcium or magnesium carbonate. The suitable pH of inoculant carrier is around 6.5–7.0.
Fine agricultural lime is used for adjusting carrier pH.
Adjusting pH of peat should be done carefully allowing time for equilibration. The reaction
between limestone and H
+
in peat will depend on particle size of both limestone and peat.
The more finely milled the ingredients the faster the reaction. Moisture content of peat is also
important to allow the reaction to occur. The amount of limestone required to change the pH
will depend on organic matter and clay content as well as buffering capacity of the peat.
After mixing peat and limestone should ideally be allowed to react for several weeks before
pH is tested. It may also be necessary to measure pH over a longer period of time. Finely
milled agricultural lime (Aglime, calcium carbonate with some impurities passing through a
150 µm mesh) is the best limestone to use to adjust pH. Builders lime is too caustic and other
lime may be too weak
The amount of lime is calculated as following:
- Suspend 10 g of carrier into 90 ml of water in a 400 ml-glass beaker
- Stir the mixture on a magnetic stirrers while monitoring the pH with the electrode of a

pH meter and gradually add lime until a pH of 6.5 has been reached
- Record the amount of lime needed to neutralize 10 g of the carrier
- Add the corresponding amount of lime to amount of the carrier. Mix well.


22
Water holding capacity: Water holding capacity of a carrier determines the amount of
liquid inoculum that can be added to the carrier. Carriers vary greatly in their water holding
capacity. Particle size, organic matter and clay content of peat will affect water holding
capacity and water potential. It is desirable to increase the water holding capacity of the peat
so that larger amounts of broth culture (and hence more cells) can be introduced to peat
before incubation.

The inherent moisture level of the carrier is determined. This is done conveniently on a
moisture balance but can use a drying oven if a moisture balance is not available. Weigh 10 g
of peat accurately on a foil or glass weighing disk and place it into the oven at 70
0
C in 48 hrs.
Check the end point of moisture loss.

Moisture content= [(W1 – W2) x 100%]/W2
W1: weight of carrier before drying
W2: weight of carrier after drying

Determination of the moisture holding capacity of the carrier. Weigh of 100 g of oven dried
carrier material into a 500 ml beaker. Add water with continue stirring until the carrier
appears to be saturated. Add additional water to produce a thin slurry. Transfer this slurry to
a pre-weighed measuring cylinder. Allow water to drain overnight, then weigh the measuring
cylinder with the contents. Give the moisture holding capacity on the dry weight basis of the
carrier. If 100 g of pre-dried carrier can hold 120 ml of water, its moisture holding capacity is

120%.

Determine the desirable amount of liquid to be added to the carrier. Prepare 6 polyethylene
bags containing 50 g of carrier. To the first bag, add the amount of water which is 5 ml less
than the carrier moisture holding capacity. If the holding capacity is 60 ml (or 120%) add 55
ml. To the next bag add add 5 ml less (50 ml). Continue until each successive bag has
received 5 ml less than the preceding one. Therefore bag number 6 will receive 30 ml. Seal
the bags and massage the bags throughout until all moisture has been absorbed and the
carrier/water mixture appear to be homogenous. Allow the six treatment to equilibrate for
two hours then open the bags and take few grams of each bag onto hand. A suitable
carrier/water mixture should feel moist, but not soggy. It should be crumble in hand and it
should not be sticky. Record the carrier/water ratio and use this information to calculate the
recommended moisture level for each carrier. The recommended moisture level is usually
given in percent calculated on the wet weight basis of the final preparation.

Amount of broth cultures added into peat varied depending on peat (Table 15). U Minh peat
was similar in water holding capacity with commercial Australian peat. In may cases high
water holding capacity of peat translates into high quality inoculants.

Moisture contents of 40 to 50% proved optimal for growth and survival of a range of
rhizobial strains prepared as peat cultures. Optimum moisture content of peat or mixtures of
peat with other ingredients should be considered (Table 16). Before injecting peat with broth,
peat must be sterilised with a moisture content of 20%. The efficacy of sterilisation should be
measured by injecting broth without bacteria and measuring growth of contaminants over
time for one month. Suspend peat and dilute as is done when counting rhizobia and spread
onto the surface of glucose peptone media. Record the dilutions at which growth occurs.

23
Table 15. pH and amount of broth cultures impregnated into moist sterilized peat
*


Peat pH Amount of broth cultures
Australia 7 65
IAS 6.5 24
Nghe An 5 50
U Minh 6 60
DakNong 6 38
Komix 1 5 35
Komix 2 5 25

Source: OPI
Australian and IAS peat were already adjusted pH with lime
moist sterilized peat
*
: 20% moisture content

Calculations based on 70 g dry peat after adjusting to 20% moisture content for sterilisation

Table 16. Treatments for measuring optimum moisture content for legume inoculants

Moisture content (%) Liquid added (mL) Volume of broth (mL)
Volume of sterile water
(mL)
40 29.5 29.5 0
50 52.5 29.5 23
60 87.5 29.5 58

Equation [1] is used to calculate moisture content of 70 g dry peat. The same equation can
be used for any quantity of peat but if the peat is moist the mass of dry peat must first be
calculated.

70 100
x
y
x
=
+
[1]

Where x is the amount of liquid added and y is the final percent moisture (eg. 50).

To adjust 70 g of dry peat to 20% for sterilisation 17.5 mL of water should be added.

An example calculation: How much liquid (eg. broth) is added to 150 g peat with 20%
moisture to get a final moisture content of 35%?

a)
Mass of dry peat

20
100
x 150 g = 30 g

150 g – 30 g = 120 g

24
b)
Moisture to add to dry peat

35
120 100
x
x
=
+


0.35(120 )
x
x
=
+

42 0.35
x
x
=
+


0.35 42xx
−

=

0.65 42x
=


64.6x
=


64.6 g moisture should be added to 120 g dry peat to achieve 35%. If peat already has 30 g
moisture then 64.6 g – 30 g = 34.6 g should be added to 150 g peat.

Table 17. Experimental design to measure effectiveness of different carriers

Moisture content (%)
Carrier
40 50 60
Peat
Peat + worm cast
Peat + coir dust
Peat + worm cast + coir dust
There should be 3 replicates of each treatment. Counts to
be done at 1 week and 1 month. Counts should be on plates
as well as plant infection tests. Two plants should be
inoculated from each of the 10
-5
and 10
-6
dilutions.

Figure 7. Counting and confirmation of viable rhizobia from contaminated peats
10
-2
10
-3
10
-4
10

-5
10
-6
Carrier suspended in sterile water (10 g in 90 mL, 10
-1
dilution)
Spread 0.1 mL on the surface of duplicate
CRYMA plates and count colonies after growth
taking note of diltuions with contamination
Prepare dilution series to 10
-6
Inoculate 2 plants
from each of the
10
-5
and 10
-6
dilutions and
check for nodules
to confirm colonies
are rhizobia on the
corresponding
plates
10
-2
10
-3
10
-4
10

-5
10
-6
Carrier suspended in sterile water (10 g in 90 mL, 10
-1
dilution)
Spread 0.1 mL on the surface of duplicate
CRYMA plates and count colonies after growth
taking note of diltuions with contamination
Prepare dilution series to 10
-6
Inoculate 2 plants
from each of the
10
-5
and 10
-6
dilutions and
check for nodules
to confirm colonies
are rhizobia on the
corresponding
plates