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| Viability of Coffee Leaf Rust (Hemileia vastatrix) Urediniospores
Stored at Different Temperatures |
| Deepak K1*, Hanumantha BT1 and Sreenath HL2 |
| 1Coffee Research Sub-Station, Chettalli, Kodagu-571248, Karnataka, India |
| 2Plant Biotechnology Division, Coffee Board, Unit of Central Coffee Research Institute (CCRI), Dr. S. Radhakrishnan Road, Manasagangothri, Mysore-570 006, Karnataka,
India |
| *Corresponding author: |
Deepak K
Coffee Research Sub-Station
Chettalli,Kodagu-571248,Karnataka,India
E-mail: deepakkumardeo@gmail.com |
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| Received June 05, 2011; Accepted July 17, 2012; Published July 20, 2012 |
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| Citation: SDeepak K, Hanumantha BT, Sreenath HL (2012) Viability of Coffee
Leaf Rust (Hemileia vastatrix) Urediniospores Stored at Different Temperatures. J
Biotechnol Biomater 2:143. doi:10.4172/2155-952X.1000143 |
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| Copyright: © 2012 Deepak K, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited. |
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| Keywords |
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| Hemileia vastatrix; Urediniospore preservation at low
temperature; Viability; Germination; Coffee leaf rust |
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| Viability of spores samples of Hemileia vastatrix, the fungal
pathogen causing leaf rust disease in coffee was checked after storing
at room temperature, -20°C and -80°C for one year. While the spore
samples lost their viability as early as 45 days at room temperature and
thus retained viability for about 120 days at -20°C. Spores stored at
-80°C retained about 52% viability even at the end of one year. The
results clearly indicated that urediniospores of Hemileia vastatrix can
be preserved at -80°C for one year and more. |
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| Coffee is the most important agricultural product in the world, being
second only to the petroleum in the global trade. India is among the
top six largest coffee producers in the world. India produced 43,72,000
bags and exported 31,45,000 bags in the year of 2008/09 (ICO, http://
www.ico.org/). Globally 65% of total coffee production comprises of
the arabica coffee. C. canephora accounts for the remaining 35%. Coffea
arabica varieties produce good coffee quality, but the genetic diversity
in the species is limited, rendering the plants highly susceptible to pests
and diseases. Coffee leaf rust (CLR) (orange rust) is the most destructive
disease of coffee caused by the fungus Hemileia vastatrix. Even thoughH.vastatrix can attack several species of the genus Coffea, it is most
devastating on C. arabica. It was first of all discovered in Lake Victoria
in Africa in 1861 and subsequently observed in Srilanka in 1868 and in
Brazil in 1970, reported from India in 1869. The pathogen has spread
to all the coffee producing countries in the world. The infection is
manifested as yellow-orange powdery lesions on the abaxial surface
of leaves, and leads to impaired photosynthesis, premature defoliation
and reduced floral initiation resulting in tremendous yield loss that may
reach as much as 70%. The urediniospores produced in these lesions
are responsible for the dispersal, survival and infection structure of the
pathogen. Teliospores are occasionally produced, germinate to produce
basidiospores. However, infection of basidiospores to coffee has not
been demonstrated and so far, no alternate host has been found. The
pathogen exists in several physiological forms or races and breaks down
the resistance in the host plant in due course by evolving new races.
While 45H.vastatrix races have been identified globally, as many as
37 races (26 designated + 11 new races) have been isolated from India
[1]. At present, the isolated and identified rust races are maintained on
susceptible plants under green house conditions. It requires frequent
re-inoculation from old leaves to new leaves in isolated chambers to
avoid cross contamination. Maintenance of large number of races
requires larger space and manpower. The infected leaves defoliated
after few days of infection which may cause loss of known races and
to re-isolate the same race requires couple of years. Hence, the study
for preservation of known rust races with high viability was optimized. |
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| The degree of resistance to rust by coffee lines depends on the
type of rust race [2]. Many studies are presently being carried out to
understand the molecular basis of the interaction between coffee and rust [3]. Parallel to this, plant breeders are putting in tremendous
efforts to generate introgressed lines that exhibit disease resistance to
different races of rust. |
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| One major requirement for studies like screening for natural
resistance in the host germplasm and screening of fungicides to control
rust outbreaks is the continuous availability of viable spores. Since,H.vastatrix is a biotropic fungus and culture of the fungus on an artificial
medium is not possible.H.vastatrix cultures are usually maintained by
repeated infection on susceptible plants under controlled conditions,
which is a tedious and laborious process. An alternative to overcome
this problem is to preserve spores under appropriate conditions.
Though there is a considerable literature available on the germination
conditions of coffee rust [4], there are only limited studies on
preservation of CLR spores [5,6]. The present study was conducted to
identify the condition for preserving the CLR spores at least for a year. |
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| Urediniospores samples of race I and race VIII ofH.vastatrix
were harvested from the mature pustules on leaves of susceptible germ
types of arabica coffee plants maintained in green house at Regional
Coffee Research Station, Thandigudi, Tamil Nadu, India. Harvested
spore samples were put in pharmaceutical grade gelatin capsules and
transported to Plant Biotechnology Division, Mysore under ambient
conditions for the study. These spore samples were used in the present
study. Viability of the spores was calculated at different intervals upto
one year after storing at dark room temperatures, -20°C and -80°C. |
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| Storage and testing the viability of spores |
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| Spore samples in 10 mg aliquots in 0.5 ml micro tubes (Tarson)
were stored at room temperature, -20°C in a deep freezer (Forma
Scientific) and -80°C in an ultra-deep freezer (Forma Scientific) in dark.
Viability of spore samples was checked before storage and at different
intervals of storage in different treatments up to one year. For checking
the viability, small aliquots of spore samples were germinated on water
agar. Spore samples stored at -20°C and -80°C were incubated in a water
bath (Grant) at 40°C for 10 minutes as suggested by Kushalappa (1989)
before plating on water agar. The spore aliquots (about 10 mg) were
suspended in 5 ml sterile reverse-osmosis grade water by vortexing for
10 seconds. 1 ml spore suspension was spread uniformly on 2% water
agar in petri plates under sterile conditions. The agar plates were sealed with parafilm (Sigma) and incubated overnight for 16 h at 23°C in dark
in an incubator (Sanyo). After incubation, the spores were observed
under a light microscope (Nikon) at 10X magnification and stored for
germination. Five replicated plates of each sample were used to assess
the germination frequency. On each plate, 100 fields of view of around
0.6 cm2 were observed randomly and the germinated/ungerminated
spores were counted (Figure 1). Germination percentage was calculated
for each replicate. Arithmetic mean was calculated from the percentage
germination value of the five replicates of each set of experiment and
the standard deviation was calculated. Viability of spores stored at
different temperatures is given in Table 1. |
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Figure 1: Germination of Hemileia vastatrix urediniospore after storing at different temperatures a, b, c: Germination of fresh urediniospores cultured for 8h,(a)
12h,(b) and 16h(c) on water agar. Swollen appresorium like structure is seen at the end of the germ tube. (D) Several viable spores have germinated after storing
at -80°C for one year indicating good viability. (E) Non-viable spore. |
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| Room temperature 25 ± 5°C |
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| The germination of fresh spores was 30% in both the races race I
and race VIII. By 15 days of storage at room temperature (25 ± 5°C),
germination percentage was greatly reduced to 94% in race I and
77% in race VIII. By 60 days onwards the germination was negligible,
although the aliquots of the same stock preserved under cold storage
were still viable. |
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| Deep freezer (-20°C) |
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| Rust race stored at -20°C showed some improvement in germination for race VIII with about 55% spores being germinated.
However, no significant change was observed in case of race I stored at
-20°C until 60 days of storage where the mean germination percentage
recorded was around 45%. By 60 days race VIII spores preserved at
-20°C had showed reduction in revival. Race I spores stored on -20°C
germination percentage was reduced completely on 210 days of
preservation whereas viability was retained in race VIII spores for one
year of preservation. |
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| Ultra-deep freezer (-80°C) |
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| After the storage at -80°C for 15 days, there was some increase in
germination percentage in both the races. Germination percentage of
spores stored at -80°C showed fluctuation over the sampled period from
21.16 to 51.98%. Although the higher viability was noticed after 15 days
for both the races at -80°C, it disappeared by 30 days in case of race I
with germination percentage falling to around 30%. A second peak of
higher viability was noticed for race-I after 60 days of storage when
the germination percentage was recorded almost 55%. Preservation
beyond 120 days showed a uniform increase in germination percentage
and was last recorded to be about 52% at the end of one year. Race VIII
spores preserved at -80°C differed from race I in their state of higher viability, which was maintained almost constant from 15 days to 60
days but fell steeply after 210 days. A second state of higher viability
was observed after 360 days when the spores recorded viability of 52%. |
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| Cold storage at both -20°C and -80°C seems to enhance germination
in both the races at least up to 15 days. This easy preservation condition
would eliminate the need of cumbersome processes like maintenance
culture on susceptible plants or constant supply of liquid nitrogen for
storage at -196°C. The results obtained herein would benefit researchers
of various fields, providing them easy preservation of rust considering
the fact that viability of coffee rust is more sensitive to temperature and
light conditions [4]. Coffee rust spores were maintained up to 150 days
with high viability in liquid nitrogen at -196°C [5]. |
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| Acknowledgements |
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| The authors are grateful to Dr. Jayarama, Director of Research, Central Coffee
Research Institute for carrying out this work. |
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| References |
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- Prakesh NS, Ganesh D, Bhat SS (2005) Population dynamics of coffee leaf rust and recent advances in rust research in India 411-442. In: Durable Resistance to Coffee Leaf Rust (Eds) Zambolim L, Zambolim EM, Varzea, V M P. Universidade Federal de Vicosa, Vicosa, Brazil.
- Rodrigues CJ, Bettencourt AJ, Rijo L (1975) Races of the pathogen and resistance to coffee rust. Annu Rev Phytopathol 13: 49-70.
- Diana F, Patricia S, Caroline A, Marie-Claude B, Anne-Sophic P, et al. (2004) Coffee (Coffea Arabica L.) genes early expressed during infection by the rust fungus (Hemileia vastatrix).Molecular Plant pathology 5: 527-536.
- De Jong EJ, Eskes AB, Hoogstaten JGJ, Zadoks JC (1987) Temperature requirements for germination, germ tubegrowth and appressorium formation of urediospores of Hemileia vastatrix. European Journal of Plant Pathology 93: 61-71.
- Kushalappa AC (1989) Biology and Epidemology. In Coffee rust: Epidemiology, resistance, and management Eds. Kushalappa, A.C. and Eskes, A.B., CRC Press. 13-80.
- Escobaro C, and Cristancho AM (2007) Estudio de metodolongias para la conservacion de urediniosporas de la roya del cofeto. Cenicafe 58: 324-332.
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