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Effect of Seed Borne Pathogens on Seed Longevity in Chickpea and Cowpea

under Storage at 25oC to – 18oC

Article  in  Seed Science and Technology · August 2006

DOI: 10.15258/sst.2006.34.1.08

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SEED LONGEVITY AFFECTED BY SEED BORNE PATHOGENS

Ahmad, Z., Ghafoor, A. and Bashir, M. (2006), Seed Sci. & Technol., 34, 69-75
© International Seed Testing Association 2006

Effect of seed borne pathogens on seed longevity in chickpea

and cowpea under storage at 25°C to -18°C
Z. AHMAD1, A. GHAFOOR1* AND M. BASHIR2

1
Plant Genetic Resources Programme (PGRP), Institute of Agri Biotechnology and Genetic Resources, National
Agricultural Research Centre, Islamabad, Pakistan (E-mail: ghafoor59@hotmail.com)
2
Plant Virologist, National Agricultural Research Centre, Islamabad, Pakistan

(Accepted November 2004)

Summary

Effect of fungal pathogen, Ascochyta rabiei on chickpea and Blackeye Cowpea Mosaic Virus (BlCMV) in
cowpea was determined on seed longevity in three different seed storage regimes (25°C, 0°C and –18°C) over
a period of ten years. Partitioning of variation in to various factors indicated less influence of BlCMV infection
for seed conservation as compared with A. rabiei. Seed viability in chickpea decreased rapidly due to infection
by A. rabiei, whereas storage at 0 or –18°C affected seed viability insignificantly in healthy seeds. Blackeye
Cowpea Mosaic Virus did not affect the initial seed germination, although storage of seeds at 25°C resulted 100
percent decline in germination after 5 years in healthy as well as contaminated seeds. The results suggested that
healthy seeds should be produced along with continuous seed health monitoring for storage in the gene bank for
future use of genetic resources. In chickpea, infection by A. rabiei increases the rate of deterioration and hence
a shorter time before regeneration is necessary in a gene bank, whereas there is little effect of BlCMV on loss
of viability in cowpea. Rapid decrease after four years at high temperature suggested that chickpea should be
preferably stored at low temperature to enhance the span for multiplication that will minimize the chance of
loosing original variability in germplasm.

Introduction

According to an estimate, more than 90% of the field crops grown in the world are
propagated through seeds, and all are attacked by devastating seed borne pathogens.
Collection, conservation, evaluation and distribution of the germplasm are the main activities
of gene banks (Kaiser and Hannan 1986). The seeds are stored preferably at 0 – 5°C in
active collections and at –18°C with low relative humidity for base collections. For orthodox
species, these storage conditions not only prolong the longevity of seeds but also the longevity
of the associated seed borne pathogens (Hewett, 1987, Kaiser, 1987, Siddiqui et al., 1983) so
the gene banks also serve as reservoirs of these pathogens (Riaz et al., 1995, Diekman, 1988,
Hampton et al., 1982). The exchange of germplasm from the gene banks during the last three
decades has been enormous (Plucknett and Smith, 1989 and Plucknett et al., 1987).

* Author for correspondence

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 Z. AHMAD, A. GHAFOOR AND M. BASHIR

Chickpea (Cicer arietinum L.) and Cowpea [Vigna unguiculata (L.) Walpers] are two
important pulse crops of the world with annual production of 6.1 and 2.5 million tons,
respectively (FAO, 2001). At present, 2,243 accessions of chickpea and 192 of cowpea
collected locally and acquired through exchange are being maintained at in the gene
bank of National Agricultural Research Centre, Islamabad, Pakistan. During our seed
testing activities, a number of accessions of chickpea (Iqbal et al., 2004), and cowpea
(Bashir et al., 2002) in the gene bank were found contaminated with Ascochyta rabiei
and BlCMV, respectively. Initially it was believed that seed contamination does not
deteriorate the longevity of seeds stored in the genebank. However Kaiser et al. (1989)
reported that germination of infected seeds was appreciably lower in lentil seed infected
with Ascochyta fabae f. sp. lentis stored at 20°C to –196°C than healthy seeds. The
information on effect of viruses is lacking in most of the crops. Therefore it is important
to investigate magnitude of deterioration in seed longevity as affected by fungal as well
as viral pathogens in various crops. Thus, the objective of this study was to investigate
the effect of fungus (Ascochyta rabiei) in chickpea and virus (Blackeye Cowpea Mosaic
Virus) in cowpea during long term storage to help the planning of future strategy for
genetic resources conservation.

Materials and methods

Seeds of Chickpea var. Paidar harvested from healthy plants and plants infected with
Ascochyta rabiei were obtained from the Food Legume Programme, National Agricultural
Research Centre, Islamabad, Pakistan. Seeds of uniform size from both the lots were
selected manually for experiment. The infected seeds were wrinkled and confirmed for
pathogen using standard techniques. Seeds with BlCMV infection were harvested from a
susceptible cultivar (Pusa Phalguni) of cowpea by growing them in the green house. The
plants were inoculated twice during growth with the inoculum isolated and maintained
on cowpea plants in an insect free green house. The identity of the virus isolate was
confirmed by Direct Enzyme-Linked Immunosorbant Assay (DAS-ELISA). The healthy
seeds of cowpea were collected from plants tested by DAS-ELISA for the absence of the
virus on the plants. For both the crops, seeds were cleaned and seeds of uniform size were
selected for inclusion in the experiment. The healthy and contaminated seeds of both the
species were dried in seed dryer at 20°C for two weeks to bring the moisture contents to 5-
7% level as recommended for conservation (Khanna and Singh, 1991). Initial germination
was determined from 400 seeds before storage at 25, 0 and –18°C, respectively. The seeds
were packed in airtight plastic bottles used in the PGRP genebank for conserving the
germplasm. Enough seeds were stored in each of 5 bottles for subsequent seed germination
tests during the experimental period of 10 years (1994-2003). Seed germination was tested
in four replicates of 100 seeds in each case using rolled paper towels moistened with tap
water and placed in an incubator at 25±2°C (ISTA, 1993). The emergence of the plumule
or radicle from the seed was considered as a criterion for indicating seed viability. Final
seed germination was recorded after 10 days. The seed germination was tested for healthy
and contaminated seed stored at different temperatures at intervals of six months.

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SEED LONGEVITY AFFECTED BY SEED BORNE PATHOGENS

Results

Table 1 presents the results of data on germination analysed for three factors, i.e., storage
temperatures (25, 0 and –18°C), seed health status (diseased and healthy) and storage
duration (ten years at an interval of six months) for both the crops. Significant differences
were observed for all the factors and their interactions, but a higher amount of variance
was attributed towards seed health status and storage duration for chickpea, whereas
storage conditions were of greater importance in cowpea. This indicated less influence of
disease in case of BlCMV for seed conservation.
Table 1. Three factors analysis of variance in chickpea and cowpea.
Source of variance df Chickpea Cowpea

Replication 3 94.6 (P≤0.000) 75.6 (P≤0.000)

Storage conditions 2 90383.6 (P≤0.000) 170184.1 (P≤0.000)
Seed status 1 140934.9 (P≤0.000) 330.3 (P≤0.000)
Storage conditions – seed status 2 6329.5 (P≤0.000) 82.6 (P≤0.000)
Storage duration 20 14063.3 (P≤0.000) 6738.7 (P≤0.000)
Storage conditions – storage duration 40 1437.1 (P≤0.000) 3090.9 (P≤0.000)
Seed status – storage duration 20 1525.1 (P≤0.000) 23.1 (P≤0.000)
Storage conditions – seed status – storage duration 40 1848.4 (P≤0.000) 18.2 (P≤0.000)
Error 375 4.9 5.12
CV (%) 3.9 3.2

The data presented in figure 1 shows that infection of chickpea by A. rabiei significantly
reduced germination at initial stage (0 day) as compared to healthy seeds. Healthy seeds
stored at 0°C showed only a slow decline in germination for the first 7 years while at
–18°C germination remained high compared to the other storage temperatures over the 10
years period. In contrast, the germination of infected seeds fell below 85% after 1 year at
0°C and 4 years at –18°C, with a rapid decline in germination after 4.5 years. As a result
germination had fallen to 0% after seven years at 0°C and 8 years at –18°C. Storage at
25°C reduced germination of healthy seeds below 85% after only 2.5 years, falling to 0%
after a period of 5 years. Infected seeds deteriorated more rapidly and germination fell to
0% in three years.
In contrast, infection of cowpea with BlCMV did not affect the initial seed germination
(figure 2). Furthermore a similar trend in germination was observed in all the three storage
regimes for diseased and healthy seeds that revealed a minimum effect of BlCMV on
seed longevity. The storage of seeds at 25°C resulted in 0% germination after 5 years in
healthy as well as contaminated seeds. The decline in seed germination of healthy and
infected seeds at 0°C was insignificant over a period of initial 7 years of storage but slow
decline was observed after that period. During 10 years of storage at 0°C germination
fell from 96% to 72%. Storage of both healthy and infected seeds at –18°C resulted in
germination being maintained at above 85% up to 9.5 years.
71
 Z. AHMAD, A. GHAFOOR AND M. BASHIR

First day
Six months
One year
One and half year
Two years
Two and half years
Three years
Thre and half years
Four years
Four and half years
Five years
Five and half years

Seven years
Seven and half years
Eight years
Eight and half years
Nine years
Nine and half years
Ten years
Six years
Six and half years

Figure 1. Effect of storage temperature 25°C (above), 0°C (middle) and –18°C (bottom) on chickpea healthy and
diseased seeds affected with Ascochyta rabiei. The dotted line indicates the required level of germination and
the viability lower to this demands multiplication.

72
SEED LONGEVITY AFFECTED BY SEED BORNE PATHOGENS

First day
Six months
One year
One and half year
Two years
Two and half years
Three years
Thre and half years
Four years
Four and half years
Five years
Five and half years
Six years
Six and half years
Seven years
Seven and half years
Eight years
Eight and half years
Nine years
Nine and half years
Ten years

Figure 2. Effect of storage temperature 25°C (above), 0°C (middle) and –18°C (bottom) on cowpea healthy and
diseased seeds affected with Black Eye Cowpea Mosaic Virus. The dotted line indicates the required level of
germination and the viability lower to this demands multiplication.

73
 Z. AHMAD, A. GHAFOOR AND M. BASHIR

Discussion

There was no significant decrease in germination of healthy chickpea seeds over three
years of storage at any of the treatment temperatures. However at higher temperature
germination declined earlier. Thus healthy seeds stored at 25°C needed multiplication
after three years, whereas at 0°C multiplication could be delayed up to seven years.
In case of some important donor parents or special landraces if diseased seed is to be
preserved, preferably it should be stored at –18°C or lower. Similar findings have been
reported by Kaiser et al. (1989) in lentil who reported insignificant decrease in viability at
low and extra low storage temperature. Seeds infected by A. rabiei lose viability quicker
than healthy seeds, an observation previously noted by Cormey et al. (1987) in lentil
seed infected by Ascochyta. It is clearly important that healthy chickpea seeds should
be produced for storage in the gene bank for future use of genetic resources. In addition
chickpea should preferably be stored at low temperature to enhance the span of time for
multiplication that will minimize the chance of loosing original variability in germplasm.
In addition it is likely that the fungal pathogen may multiply even at temperature lower
than –18°C (Kaiser et al., 1989).
Unless special precautions are taken to prevent the introduction of the pathogen on
imported seeds, it is possible that the fungus will survive in infected seeds over extended
periods of time when stored at ultra-low temperatures. In this way, gene banks could
become a repository of different isolates and/or new pathotypes of the pathogen that could
be introduced into a country where Ascochyta blight does not occur or virulent pathotypes
of the pathogen are not present. Singh et al. (2003) also suggested conducting seed health
status prior to storage for conservation that will help in discarding heavily infected seed
lots and will ensure high seed viability for longer period.
In cowpea, BlCMV infection had little effect on germination in all the temperature
regimes although viability of both in healthy and diseased seeds was affected over a period
of time, i.e., 2 – 3 years at 25°C, 6 years at 0°C and 9 years at –18°C. As a result the time
to multiplication was not affected by BlCMV infection particularly at low temperature.
The implication of these results is that only disease-free seed or seed with a very low
level of infection should go for long-term conservation. This necessitates conducting seed
health test prior to storage for conservation, which will help in discarding heavily infected
seed lots and will ensure high viability of seed for longer period of time.

Acknowledgements

We acknowledge the cooperation of Food Legume Programme, National Agricultural

Research Centre for providing seed of chickpea and cowpea varieties. Technical assistance
by Dr. N. Murata and Dr. T. Mituseda is appreciated. This work would have not been
possible without financial and technical support of Japan International Cooperation
Agency (JICA) that is highly acknowledged.

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 SEED LONGEVITY AFFECTED BY SEED BORNE PATHOGENS

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