Project Title: Safe food for the poor: Detection and management of toxigeneic Fusarium

associated with sorghum grain mold complex [This is a good example of a title with a
simple first part, and a scientific second part. I like it.]
Principal Investigator
Name: Rajan Sharma

Designation: Scientist, Cereals Pathology

Institute: International Crops Research Institute for the Semi-Arid Tropics

Address: ICRISAT, Patancheru PO. Andhra Pradesh, PIN: 502 324

Co-Investigator
Name: Ram P. Thakur

Designation: Principal Scientist

Institute: International Crops Research Institute for the Semi-Arid Tropics

Address: ICRISAT, Patancheru PO. Andhra Pradesh, PIN: 502 324

Co-Investigator
Name: Varsha Wesley

Designation: Scientist

Institute: International Crops Research Institute for the Semi-Arid Tropics

Address: ICRISAT, Patancheru PO. Andhra Pradesh, PIN: 502 324

Co-Investigator
Name: Rajeev Varshney

Designation: Senior Scientist

Institute: International Crops Research Institute for the Semi-Arid Tropics

Address: ICRISAT, Patancheru PO. Andhra Pradesh, PIN: 502 324

Duration: Three years

Likely donor: DST/DBT, Govt. of India
Type of funding: Special project
Total Cost (Rs): 52,01,680

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[A general comment: This concept note includes many scientific terms and ideas; these
are fine for sending to a ‘science’ donor like the Dept of Biotech in India. If you send
this CN to a development donor, however, you will need to re-write for a non-science
reader. Because I am not a scientist, I cannot judge the adequacy or value of the research
you propose. You need to have this aspect of your CN reviewed by a supervisor, or other
people at ICRISAT.]

Summary
This proposal requests Department of Biotechnology, Govt. of India to provide funds (Rs
52.02 lakh) to International Crops Research Institute for the Semi-Arid Tropics
(ICRISAT) for initiating a project on identification of different species of Fusarium
associated with sorghum grain mold complex in India and to determine their toxin-
producing potential. The potential toxigenic strains/species thus identified will be used for
screening elite sorghum lines for resistance to mycotoxins. The project will take three3
years and 1.0 person years each of ICRISAT staff and a research scholar. This is not clear.
You name four people on page one. Will each of the four spend one year over the three
years, for a total of four person years? Please rephrase to make the exact personnel input
levels clear. This information should also be in a footnote in the budget.
The need for the project is pressing as several species of Fusarium involved in grain mold
complex produce mycotoxins, such as fumonisins, moniliformin, trichothecenes,
fusaproliferin, etc. These mycotoxins are harmful to human and animal health and
productivity. [You could make this more dramatic by giving some details of the harm
these mycotoxins do...!] Scientists at ICRISAT are anxious to produce the desired results
as soon as possible. To achieve this, molded grains of sorghum will be collected by
ICRISAT staff along with NARS partners from different locations in India. Pure cultures
of at least 100 Fusarium isolates will be established to identify different species through
gene sequencing. Isolates representing different species of Fusarium will be screened for
their ability to produce mycotoxins to select at least five5 toxigenic strain/species for
screening sorghum for genetic resistance. Finally, a reference set of Fusarium species
associated with grain mold complex in India will be developed and sorghum lines
resistant to toxigenic Fusarium made available for use by NARS partners.

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The project builds on previous work by scientists at the Central Food Technological
Research Institute, Mysore, India in collaboration with ICRISAT. F. proliferatum was
identified as major trichothecenes producing species among 11 Fusarium isolates from
sorghum analysed for toxin production and species identification. ICRISAT is ideally
suited to conduct the proposed project because it has excellent greenhouse facilities for
effective screening of sorghum genotypes against mold fungi and a well equipped
Applied Genomics Laboratory with the basic infrastructure for gene sequencing.
ICRISAT is also maintaining about 100 isolates of Fusarium from molded sorghum
grains collected from diverse locations in India. Usually you don’t need an introduction
for a concept note, but I quite like this one, since it is quite short and helpful.

Rationale/Need
Grain mold is a major biotic constraint in the production marketing and utilization of
grain sorghum. The term “grain mold” is used to describe the diseased appearance of
sorghum grains resulting from infection by one or more pathogenic/saprophytic fungi.
The disease is particularly important on improved, short and medium duration sorghum
cultivars that mature during the rainy season in humid, tropical and subtropical climates.
Several fungal spp. of the genera Fusarium, Curvularia, Alternaria, Phoma, Bipolaris
and Colletotrichum have been reported to be associated with grain mold. Of these,
Fusarium spp. are dominant within the grain mold complex. Some species of Fusarium
(F. andiyagi, F. proliferatum, F. thapsinum, F. sacchari and F. verticillioides) involved
in grain mold complex produce mycotoxins, such as fumonisins, moniliformin,
trichothecenes, and fusaproliferin. etc.[Try not to use ‘etc’ in a concept note or proposal –
it always looks lazy!] The secondary invading Aspergillus spp. may produce aflatoxins in
the contaminated grains as well. These toxins reduce the quality of grains as food/feed
sources as well as the value of grain in the market. These toxins are associated with a
variety of human and animal health problems including acute toxicity, increased
incidence of cancer, inhibition of normal growth and development, immune suppression
and increased disease susceptibility, increased risks of birth defects and reduced
nutritional quality of resulting grain. Risk analysis studies have indicated that when these

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molds affect staple food grains such as sorghum and maize, the significance to human
and animal health is considerable because their quantum of consumption is higher than
that of other foods.
The first report of fumonisins toxicity to human and poultry came in 1998 from the
Deccan plateau of India. An outbreak of food poisoning, characterized by abdominal pain
and diarrhea, attributed to the ingestion of fumonisins-contaminated maize and sorghum,
hads been reported from several villages in India. The disease was observed only in
adults who were consuming the molded grains. The affected people mostly belonged to
lower socioeconomic groups, such as marginal farmers and landless agricultural
labourers who did not have access to other staples. Similarly, an outbreak of
trichothecene mycotoxicosis associated with the consumption of mold-damaged wheat
hasd been reported from Kashmir Valley in India. Although mycotoxins have been
detected from various food grains infected with Fusarium, however, the information
regarding the toxin- producing species of Fusarium involved sorghum grain mold
complex in India is not yet available. Since several species of Fusarium have been
reported to be associated with sorghum grain mold complex, it is essential to identify
more precisely the toxigenic species of Fusarium and genetic resistance in sorghum
against potential toxigenic strains. It is in this context perspective that this project is
being proposed.
Scientists at ICRISAT have invested considerable time and efforts in towards
understanding the biology and epidemiology of fungi associated with the grain mold
complex, and have developed well-refined screening techniques to identify genetic
resistance in sorghum against grain mold. In addition, ICRISAT has a highly experienced
team comprising consisting of of plant pathologists, a molecular biologist and sorghum
breeders available to work on the project.
I like the fact that you have included “what has already been done”, so that readers know
you understand the background to your work.

Project goal
The overall goal of the project is to increase food safety in sorghum-consuming human and
animal populations in India and elsewhere. [If you want to go beyond India, then please

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name some or all of the other countries where your work would apply in your introduction
and rationale’needs sections. This would be particularly important for a development
donor.]

Objectives
The general objective of this project is to identify and characterize all the toxin-producing
Fusarium species associated with grain mold in India and identify genetic resistance in
sorghum against these. [Note: To make this objective SMARTER, I have added “all the”
to the sentence above. Only accept this suggestion, if it is true. If you are only going to
identify two or three or four species, then say so.]

Specific objectives are:

1. Identification of Fusarium species associated with grain mold complex in India
by partilly sequencing at least 100 isolates for three genes, β-Tubulin, α-
Elongation factor and Calmodulin, and AFLP analysis.

2. Screening of 100 isolates of Fusarium for fumonisins and trichothecenes

production through HPLC and GC-MS, and selection of at least five5 toxigenic
strain/species for screening sorghum for genetic resistance.

3. Screening of 50 sorghum lines under field conditions and 15-20 selected lines in
the greenhouse for genetic resistance to toxigenic strains of Fusarium.
[I like these objectives – they are SMART – and it will be easy, at the end of the project,
to see if each objective was achieved.]

Project Activities/Milestones
Activity Milestone
Determine Year 1
frequency of 1. At least 100 samples of molded sorghum grains collected and
Fusarium in frequency of Fusarium in relation to other pathogenic fungi
molded grains involved in complex determined.

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and identify 2. At least 100 isolates partially sequenced for three genes, β-
different Tubulin, α-Elongation factor and Calmodulin for sequence based
Fusarium species identification of Fusarium species.
involved in grain Year 2
mold complex 1. Mating types determined using mating type specific primers.
(Year1 & 2)
Identify toxin- Year1
producing strains 1. At least 100 isolates screened for their ability to produce
of Fusarium fumonisins and trichothecenes.
(Year 1, 2, 3) Year 2
1. Toxigenic strains of different species of Fusarium identified.
Year 3
1. DNA markers specific to at least two toxigenic species of
Fusarium developed.
2. Reference set of Fusarium species associated with grain mold
complex in India developed.
Identify genetic Year2
resistance in 1. Aat least 50 morphologically diverse sorghum lines screened
sorghum lines to in a grain mold nursery following artificial inoculation by highly
toxigenic toxigenic strain of Fusarium
Fusarium (Year 2 Year 2
& 3) 1. Selected 15-20 sorghum lines from field screen screened against
at least five5 toxigenic strain/species of Fusarium in greenhouse
[I like the idea of a chart, but this one is difficult to read. It would be easier to read perhaps,
if it were single-spaced, and fit on a single page. See if you can improve it in the next draft.]

[Note: I follow the rule that all numbers under 10 should be spelled out. All numbers 10
and over are written in numbers. This rule does not apply to Year 1, Year 2, Year 3, which
are specific times, with capital letters!]]
Work plan:
Identification of Fusarium spp. associated with grain mold complex
During Yyear 1, grain mold samples from different susceptible lines in the sorghum grain

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mold resistance stability nursery (SGMRSN) conducted at different locations will be
collected by ICRISAT scientists and NARS partners. SGMRSN is a collaborative nursery
project, managed collaboratively by between ICRISAT and the All India Coordinated
Sorghum Improvement Project (AICSIP) and is conducted at 5-6 locations every year.
The fFrequency of Fusarium associated with molded grains of sorghum at different
locations in India will be determined and pure cultures of at least 100 isolates established
at ICRISAT. DNA will be isolated from these isolates and used for AFLP, sequencing
phylogenetically important genes, identifying mating types and testing for the presence of
genes that are important in mycotoxin biosynthesis. These isolates will be partially
sequenced for three genes, β-Tubulin, α-Elongation factor and Calmodulin for sequence
based identification of Fusarium species and also analysed through amplified fragment
length polymorphism. During 2nd year, the isolates will be tested for mating type alleles
[what are these???} using specific primers to determine frequency of mating type loci
across locations. The isolates will also be tested for the presence of genes that are
important in mycotoxin biosynthesis using gene specific primers.

Identification and characterization of toxigenic strains of Fusarium

At least 100 isolates will be screened during Yyear 2, for their ability to produce
fumonisins and trichothecenes through HPLC and GC-MS. Five toxin- producing strains
will be selected for screening sorghum genotypes for the management of this disease
through host plant resistance. Attempts will be made to identify DNA markers specific to
at least two species of Fusarium associated with grain mold complex during yYear 3.
Finally, a reference set of Fusarium species associated with grain mold complex in India
will be developed for use by NARS partners.

Screening sorghum lines for genetic resistance to toxigenic strains of Fusarium

During Yyear 2, at least 50 morphologically diverse sorghum lines that have been
identified as resistant to grain mold complex (species of Fusarium, Curvularia,
Alternaria and Phoma) will be screened under field conditions at ICRISAT following

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artificial inoculation with highly toxigenic strain of Fusarium. During Yyear 3, 15-20
selected lines from the field screen will be further tested against five5 different
strains/species of Fusarium to identify sorghum lines with stable resistance to toxigenic
strains. The resistance sources thus identified will be made available to breeder at
ICRISAT as well as NARS partners to develop sorghum cultivars resistant specifically to
toxigenic Fusarium and grain mold in general.

Outputs
1. Toxigenic Fusarium spp. associated grain mold complex identified and a reference
set of Fusarium strains made available to NARS partners
2. Sorghum lines resistant to toxigenic strains of Fusarium spp. associated with grain
mold complex identified and made available to breeders for use in sorghum
breeding program
[Will you write this work up in a paper? Will you give this paper at a conference? If
so, these might be additional outputs of your project.

Gains Expected
[When do you expect these gains? This is the impact of your work, and very important, if
you rewrite this concept note for a development donor. You will need to say a lot more
about the impact of your work on the health of humans and animals for this sort of donor –
for instance, when will the gains be evident, how many people will be affected, what sort of
people and animals will benefit, etc., etc. ]
 Decreased qualitative and quantitative losses in sorghum grain production and
thus increased farm income.
 Reduced mycotoxin contamination of sorghum grains, thus reduced impact on
health of humans and domesticated animals.

Users
Initially, tThe direct users of research outputs of this project will be researchers, particularly
breeders at ICRISAT and NARS partners. However, eventually, initially and farmers and

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consumers will be the real beneficiaries, enjoying safer food. eventually. Mycotoxins have
implications with human and animal health and productivity, and global sorghum trade.
Decreased mycotoxin contamination will increase the health of humans and domesticated
animals. [Even for the Indian donor, I think you need to say a good deal more here,
quantifying the numbers of beneficiaries, and describing them. It is likely that large
numbers of very poor people would benefit from healthy sorghum. This is the place to
say so!]
Logical Framework Matrix

Narrative Objectively verifiable Means of Important

summary indicators verification assumptions
Goal Increased food At least 5 Fusarium Project Resistance to
safety in sorghum resistant sorghum lines report, Fusarium
consuming areas of included in breeding ICRISAT available in the
semi-arid tropics program archival sorghum
report genotypes
Purpose Identify toxin 1.Five toxigenic Project Fusarium
producing Fusarium strains/species of report, continues a
species associated Fusarium identified Publications major pathogen
with grain mold in 2.About 50 elite in sorghum
India and genetic sorghum lines screened grain mold
resistance in for resistance to complex
sorghum to the Fusarium
pathogen
Outputs 1.Toxigenic 1.Reference set of Research Project is well
Fusarium species Fusarium strains made and project managed
identified available to NARS reports
2.Sources of partners
resistance to 2.At least 5 lines of
Fusarium in sorghum resistant to
sorghum identified Fusarium identified

Activities Determine 1.Pure cultures of at Research The project is

frequency of least 100 isolates and project well designed,
Fusarium in molded established and reports, funds come on
grains and identify analysed for Publications time, qualified
different Fusarium fumonisins and people available
species involved in trichothecenes on time.
grain mold complex 2. Selected isolates
and resistance in partially sequenced for
sorghum to β-Tubulin, α-
pathogenic Elongation factor and
Fusarium Calmodulin genes for
species identification

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 3. About 20 sorghum
lines screened against 5
toxigenic strains and
sources of resistance
identified

BUDGET PARTICULARS
Budget (In Rupees)

[I think you should improve this budget by providing more details in a footnote for each
line item. For instance, what equipment will you buy for 5,00,000? How much time of
what sort of scientists is the donor buying each year? Why is this amount the same each
year? Won’t the people get any raises? Each line item raises questions like that, which
your budget should answer. Please work with someone from PDMO and/or Finance
Office to get the right level of detail.]
Overall I think this is a modest budget. Maybe too modest. By my calculations, you are
asking for the equivalent of $100,000 over three years to do something with potentially
very significant outcomes. Are you sure you can achieve your outputs with this budget?
Is there an ICRISAT contribution?
S. No. Item Year 1 Year 2 Year 3 Total
1 Equipment 5,00,000 5,00,000
2 Staff (SRF) 3,74,400 3,74,400 3,74,400 11,23,200
3 Screening for mold resistance 3,00,000 3,00,000 6,00,000
4 Fine chemicals for gene 4,50,000 2,00,000 4,50,000 11,00,000
sequencing/AFLP
5 Mycotoxin estimation 4,00,000 4,00,000 8,00,000
through HPLC
6 Travel 1,00,000 75,000 75,000 2,50,000
7 Office supplies 50,000 50,000 50,000 1,50,000
8 Overhead (15%) 2,81,160 2,09,910 1,87,410 6,78,480
Grand Total 21,55,560 16,09,310 14,36,810 52,01,680

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