Journal of Agricultural Sciences
Vol. 52, No 1, 2007
Pages 17- 31
UDC: 633.491:631.24
Original scientific paper
THERMAL CONTROL OF SOME POST-HARVEST ROT
PATHOGENS OF IRISH POTATO (Solanum tuberosum L.)
Abiodun Olusola Salami1 and Olumide Omololu Popoola2
Abstract:Thermal control effect on the incidence of some post-harvest rot
pathogens of Solanum tuberosum (potato) was investigated in this study. Three
cultivars of potato tuber whose local names are, Patiska, Mai Bawondoya and
Nicola were used for the study. Five pathogenic fungi viz: Botryodiplodia
theobromae, Fusarium redolens, Fusarium oxysporum, Penicillium sp. and
Rhizopus oryzae associated with post harvest storage rot of root-tubers, were
isolated from diseased potatoes. Among the three species of potatoes used in the
study, Patiska was found most resistant followed by Mai Bawondoya, while
Nicola was the least resistant. Increase in substrate (i.e. soluble starch or CMC)
concentration enhanced a proportional increase in mycelial growth and in the
amount of extracellular enzymes produced. Some of these test pathogens were
found to produce cell wall degrading enzymes (i.e. amylase and cellulase).
Preferential utilization of carbohydrate sources was established in this study
based on the growth of test pathogens. Growth on potato broth medium was
highest followed by growth on Cocoyam and Sweet potato broth media and least
on Cassava broth medium. Growth of the test pathogens on carbohydrate sources
was found at variant.
The use of hot water treatment at different temperatures was found to
significantly reduce post-harvest fungal populations on the surface of root-tubers.
The efficacy of blanching in hot water at 60˚C was significantly higher than that
of blanching in hot water at other temperatures. The control method adopted in
this study showed that the problems of potatoes’ rot disease in storage (especially
by the peasant farmers) can be eradicated by thermal treatments without reducing
the quality of the Irish tuber.
Key words: potato tuber, diseases, thermal treatment.
1
2
Ibadan
Abiodun Olusola Salami ,Plant Science Department, Obafemi Awolowo University, Ile-Ife
Abiodun Olusola Salami ,Botany And Microbiology Department, University of Ibadan,
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Abiodun Olusola Salami and Olumide Omololu Popoola
Introduction
After poverty itself, the third world’s most pressing problem is food scarcity.
The International Food Policy Research Institute (1977) predicted a bleak future
for the world’s food situation. The food deficit in the developing market
economics is projected to rise from 12 million metric tons in 1975 to 70-85
million by 1990, Asia accounts for 40% of the total projected, North
Africa/Middle-East about 25%, Sub-Saharan Africa over 20%, and Latin
America over 10%”. Equally alarming is the rate of some 15 million of the
world’s Children who die each year of malnutrition and related diseases (FAO,
1997). Most of this mortality is concentrated in the developing countries, which
are said to home about 75% of the world’s population. Even where the “green
revolution” has made its mark, many children are malnourished (V i l l a r e a l ,
1982). This is because their diets are largely based on cereal grains, cassava and
bananas; all starchy and low in protein. It has been established that a child’s
small stomach cannot hold enough of such food to provide the protein the body
needs let alone the necessary vitamins and minerals (V i l l a r e a l , (1982); FAO,
(1997). In view of this, it is imperative that a solution is found which will check
the besetting problems of low production, post-harvest storage deterioration/spoilage
and dependence on crops of relatively low nutritional value.
In the quest for food and the struggle for human survival, Irish Potato has
historically played important role and is suitable in addressing the problems
previously highlighted. This is due to their yield per unit area (hectare) per unit
time and their nutritional value (i.e. ratio of protein to carbohydrate) (T e w e , et
al., 2003)). African farmers are faced with several constraints in the production
of food and cash crops. Some of these constraints are poor soils, poor farm
practices, use of local varieties, land tenure and damages by diseases and pests
(FAO, 1997). Irish potato tubers like many other crops are attacked by various
pathogens which cause diseases in them. Such pathogens are fungi, bacteria and
viruses. The greatest of these constraints is that of post-harvest spoilage of farm
produce, out of the several tons harvested, just a fraction is utilized while most of
the amount is lost to post harvest diseases especially rot diseases. (A l e x a n d r a t o s ,
1995). These pathogens cause great losses and reductions in the value of these
crops but with a good system of control this can be eradicated. Pathogenic fungi
associated with post-harvest Irish potato tuber rot are Alternaria solani (early
blight). Rhizogospora subtranea (Powdery Scab), Fusarium roseum and
Fusarium solani (fusarium rot disease), Helminthosporium solani (skin
blemishes disease), Colletotrichum atramentarium (black rot disease),
Aspergillus niger, Pythium ultimum (Pythium tuber rot disease), Phytophthora
erythroseptica (Pink rot disease), and Phytophthora infestans (late blight disease)
(C l a r k and M o y e r , 1988). The mechanisms by which diseases are produced
Thermal control of rot pathogens on potato
19
vary considerably with the causal agents and sometimes with the plant. Parasitic
diseases commonly arise as a result of an interchange of metabolites between a
parasite and the invaded host plant (A g r i o s , 1998). This is because enzymes are
the first substances secreted by the pathogen on establishing contact with the
surface area of their hosts. Many plant pathogenic and non-pathogenic organisms
are capable of producing an array of cell-wall-degrading enzymes.
Biological control methods encompass the utilization of organisms other
than man (C a m p b e l l , 1989). This involves the selection and breeding of plants
for resistance to particular pathogens or the use of other microorganisms that are
either antagonistic to the pathogen or parasitize the pathogen itself (A g r i o s ,
1998; W h i p p s and M c Q u i k e n , 1993; K u k i k , 1995). Although, the use and
breeding of resistant varieties (Biological control method) is the oldest, cheapest
and overall best means of controlling plant diseases, some are not economically
feasible. The use of physical agents, such as temperature (high or low) and
various types of radiation in controlling plant diseases (A g r i o s , 1998) is another
means of control that has not been adequately explored and information on its
use suggest great promise for future development. Hence this study shows the
effect of thermal method of control application on the incidence of some post
harvest rot pathogens of Irish potato tubers.
Materials and Methods
Survey and Collection of Sample
Six local markets Bodija, Oje, Sabo, Ojoo, Dugbe (in Ibadan, Southern
Nigeria) and Bukuru (in Jos, Nothern Nigeria) were visited to collect both
healthy and diseased samples. All infected tubers were inspected for rotted areas
and were stored in clean polyethylene bags. All samples were brought to the
Laboratory for further analysis.
Preparation and Sterilization of Culture Media
Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) (Difco or
Oxoid) were used for the isolation and cultivation of different fungi generally
associated with Post-Harvest spoilage/deterioration of Irish potatoes.
Isolation of Associated Fungi
Diseased samples were surface sterilized with 1% sodium hypochlorite
solution for 1 minute, rinsed in five successive changes of sterile distilled water
and then blotted dry. These tubers were cut aseptically from healthy region to
necrotic region. Discs of about 4mm diameter were made from the advancing
20
Abiodun Olusola Salami and Olumide Omololu Popoola
edge of the rot as well as from the middle of lesions and plated on media (PDA
and MEA) into which 0.1% streptomycin was added to suppress bacterial
growth. These were incubated at room temperature i.e. 28+2°C for the growth of
organisms. The organisms were to grow for 96 hours after which they were subcultured to obtain pure cultures. The pure fungi cultures were placed on slants in
Mac Cartney bottles and stored in a refrigerator for further use.
Identification of Associated Fungi
Using a sterile inoculating needle, small portions of each mycelia colony
were aseptically taken and placed on clean microscope slides and teases in a drop
of lacto phenol cotton blue. Fungi species that developed were identified with
the aid of a compound microscope, by reference to compendium of soil fungi,
D o m s c h et al., (1980), B a r n e t t and H u n t e r (1972), and by comparing with
identified species of the same genera in the microbial collection of the
Department of Botany and Microbiology, University of Ibadan, Nigeria.
Establishment of Pathogenicity of Isolated Fungi
All fungi isolates were tested for their ability to induce rot in healthy tubers.
The healthy tuber surfaces were swabbed with cotton wool soaked in 1% sodium
hypochlorite and washed thrice with sterilized tap water. Holes were dug using
sterilized 5mm cork borer and the plug was pulled out. 3mm mycelial discs of the
test pathogen were placed at the bottom of the hole. Portions of the plugs were
cut off to compensate for mycelial disc thickness before replacing in the hole and
the wound sealed with vaseline to prevent extraneous infection. The controls
consisted of sterile PDA discs of 3mm wells made on healthy tubers. The tubers
were incubated in desiccators for 2,4,6,8 and 10days respectively at 280C +20C.
All treated tubers were prepared in triplicate. After a period of 10days, the tubers
were cut across by means of sterilized scapel along the plane of inoculation and
the percentage rot was assessed using the formula below according to A j i b o y e
(2004):
Rotted surface Area x 100
Total surface Area
1
Effect of CMC concentration on mycelia growth of the test pathogens:
Carboxyl Methyl Cellulose (CMC) medium was prepared at various
concentrations of 0, 0.25, 0.5, 0.75 and 1.0%. Each medium was dispensed
measuring 30mls of each into 150-ml flasks. After sterilization, the flasks were
inoculated and incubated at 300C for 6 days. The cellulase activity of the filtrates
was then determined at 540nm using an SP 600 Spectrophotometer
Transmittance. The pH of the filtrates was also measured.
Thermal control of rot pathogens on potato
21
Effect of soluble starch concentration on mycelia growth of the test pathogens:
A soluble starch medium for enzyme synthesis was prepared with various
concentrations of starch, 0, 0.25, 0.5, 0.75 and 1.0%. Each medium (pH 6.9) was
dispensed 30mls each, into 150ml conical flasks. Five flasks of each
concentration were sterilized, inoculated and incubated at 300C for 6days. The
mycelium produced in each flask was then filtered in pre-weighed filter paper,
dried in an oven at 850C for 24hrs, cooled in a desiccator and weighed. The
amylase activity of each filtrate was determined at 300C. The pH of the culture
filtrates was also measured. Each filtrate was tested for the presence of reducing
sugar by boiling 2 ml of the filtrate together with 2ml of Benedict’s solution for
5min.
Effect of different carbohydrate sources on mycelia growth of fungal isolates
The effects of different carbohydrate media were tested on the mycelia
growth of the fungi and enzyme activities. The carbohydrate sources used were:
− Cocoyam;
− Sweet Potatoes;
− Irish potatoes;
− Cassava.
Extracts of the different carbohydrate sources were prepared by boiling 250g
each of the carbohydrate sources for 1 hour in 1 litre of distilled water. The
supernatant was collected into 1 litre-measuring cylinder and made up to 1000ml
with distilled water. Each extract was supplemented with 20g glucose. The
mixture was then dispensed into each of the 30 and 150ml conical flasks; the
process was repeated for the other broths. All flasks and contents were
autoclaved at 1210C for 15 minutes; after the content had cool, 5mm mycelial
disc of one of the test pathogen was used to inoculate 3 flasks of Cocoyam
Dextrose Broth (CDB). The same process was repeated for the rest of the test
pathogens for CDB. The above procedures were carried out for the rest of the
broth media. All flasks were incubated at 300C for 7 days. At the end of
incubation period, the mycelia mats were harvested unto pre-weighed filter
papers respectively and oven dried at 800C for 24 hours. They were then cooled
in desiccators and weighed. The dry weight of mycelia mats was obtained by
subtracting the weight of filter paper from total weight. The filtrates were
collected for enzyme assays as earlier described.
Determination of the thermal Level
Hot Water Treatment:
The root tubers were blanched in 25 Litres containers containing 20 Litres of
hot water at 28, 60 and 100°C for 25 minutes. The water was changed several
times to prevent inoculum build up. The root-tubers were subsequently air dried
22
Abiodun Olusola Salami and Olumide Omololu Popoola
and stored in well aerated storage sheds with temperature ranging between 12°C
to 18°C. The root-tubers were well spread out. Tubers to be dehydrated were
placed in boxes and sun dried for 5 days.
Results and Discussion
Incidence of Pathogenic Infection on Irish potatoes tubers
Ten fungal isolates were cultured from the rotted Irish potatoes. A total
number of five (5) properly identified fungi species were randomly selected for
use. Those selected are Botryodiplodia theobromae; Fusarium redolens;
Rhizopus oryzae; Fusarium oxysporum; and Penicillium sp.
Key
Ro = Rhizopus oryzae,
Fr = Fusarium redolens,
Bth = Botryodiplodia theobromae,
Fo = Fusarium oxysporum
Psp. = Penicillium sp.
Level of Susceptibility of potato tubers to the Different Pathogens isolated
All fungi isolated from rotted Irish potatoes were pathogenic at varying
degrees to healthy Irish potatoes when re- inoculated into them (Plates vi-xx).
Fusarium redolens and Botryodiplodia theobromae were the most aggressive and
virulent followed by Rhizopus oryzae and Fusarium oxysporum while
Penicillium specie was the least pathogenic (Fig.1).
Fig. 1. - Level of Susceptibility of Irish potatoes to Different Pathogen
Thermal control of rot pathogens on potato
23
Mycelia Dry Weight
The mycelia dry weight of all the test pathogens increased gradually with
incubation period when cultured on CMC except Fusarium oxysporum which had
reduced dry weight values on the 8th day. Also, Penicillium sp. had the highest
mycelia dry weight followed by the Rhizopus oryzae, Botryodiplodia
theobromae, Fusarium redolens and Fusarium oxysporum when cultured on
soluble starch medium (fig.2) while Fusarium redolens had the highest mycelia
growth followed by Rhizopus oryzae, Fusarium oxysporum, Botryodiplodia
theobromae and Penicillium sp. in cellulose basal medium (fig.3).
Fig. 2. - Mycelia dry weight of test pathogens in soluble starch culture filtrate
See Fig. 1 for the key
Fig. 3. - Mycelia dry weight of test pathogens in CMC culture filtrate
See Fig. 1 for the key
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Abiodun Olusola Salami and Olumide Omololu Popoola
Effects of Increased Substrate (Starch or CMC) Concentrations on the
Mycelia Growth and Enzyme synthesis
Both mycelia growth and enzyme synthesis increased as substrate
concentrations increased (fig.4-7). This shows that Mycelia growth and enzyme
synthesis are influenced by the concentration of the substrates. Penicillium sp.
had the highest mycelia growth and enzyme synthesis followed by
Botryodiplodia theobromae, while it was found least in Fusarium oxysporum
when increased starch concentration was considered. However, in increased
concentration of CMC, Fusarium redolens had the highest followed by Rhizopus
oryzae and found least in Botryodiplodia theobromae.
Fig. 4. - Effects of increased Starch concentration on Mycelia growth
Fig.5. - Effects of increased CMC concentration on Mycelia growth
Thermal control of rot pathogens on potato
Fig. 6. - Effects of increased soluble starch on enzyme synthesis
Fig. 7. - Effects of increased CMC concentration on enzymes synthesis
25
26
Abiodun Olusola Salami and Olumide Omololu Popoola
Effect of Different Carbohydrate Sources on Mycelia Growth of Fungi Isolates
Growth of the test pathogens was observed on all Carbohydrate sources used
in this study. The highest growth of the test pathogens was found on potato
followed by Cocoyam, Sweet potato while it was least on Cassava (fig.8).
Overall, Botryodiplodia theobromae recorded the highest growth followed by
Fusarium redolens while Rhizopus oryzae recorded the least growth except on
Irish potato source where Fusarium redolens was found highest (fig.8).
Effects of Thermal Control/Treatment on Root-tubers
At 28 ºC, 8%, 28%, 60% and 96% of the tubers had rotted in 2, 4, 6 and 8
weeks respectively while at 60 ºC, 4% of potatoes rotted in 14 weeks (fig. 9). At
100ºC, all the root-tubers rotted under a week (fig. 9). However, when the tubers
thus treated, were sun dried, 12%, 32%, and 44% of potato rotted in 10, 12, and
14 weeks respectively (fig. 9).
Fig. 9. - Thermal treatment effect on root tubers of Irish potatoes
Losses from Post-harvest disease of stored potatoes are of great economic
importance to nations and they are warring against food security in the
developing world (S w a m i n a t h a n and S a w y e r , 1982). These diseases also
occur during different seasons of the year, thus, potatoes are available for
consumption only during part of the year due to inadequate storage. Reduction of
post-harvest food losses, which are particularly high in perishable crops like
potatoes in developing countries, is important not only from an obligation to
avoid waste but because the cost of preventing food losses in general is
considered to be lesser than producing a similar additional amount of food of the
Thermal control of rot pathogens on potato
27
same quality (B u r t o n and B o o t h , 1982). Considering the number and
virulence of the organisms isolated from the root tubers of the Irish potatoes, the
importance of plant and pathogen interaction in food production and in postharvest management cannot be over emphasized (A j i b o y e , 2004). Of all the
organisms isolated from diseased potato root tubers in this study, five have been
found to cause spoilage of root tubers in storage facilities. The ability of these
pathogens to colonise a wide range of plant materials, shows their ubiquity and
non- host specific nature. In this study, Fusarium oxysporum had the highest
frequency and it occurred on all potatoes types. This can be attributed to its nonhost specific nature. This is in line with the findings of A n d e r s o n , (1985);
T h e r b e r g e , (1989); T e l l a , (1991); and A d e w o l u (1999). It has been
observed in this study again that pathogenic organisms establish their contacts
with their hosts by utilising enzymatic substances. This is due to the fact that all
the test pathogens isolated in this work had increased mycelia weight and growth
when cultured on different carbohydrate and carbon sources. This has also been
discovered by P a l m e r (2001) and O k o l o et al. (1995). These organisms were
also found to respond to increased concentrations of culture filtrates used in this
study, which they used to synthesize amylase and cellulose which are cell wall
degrading enzymes. R a b i n o r i c h et al. (2002) and S a l a m i (1999) also
discovered this in their studies. Also discovered in this study is the fact that
infection occurs as a result of wound created on the tuber, this is because
wounded tuber rotted easily more than the unwounded tuber. Thus, tubers should
be handled carefully during harvest to prevent wounding (R a b i n o r i c h et al.,
2002).
Hot water treatment has been proved to be very effective for preventing the
inocula of all the organisms isolated from Irish potato in this study without
injuring them. This has been noted by S h e r w o o d (1970) and P u l l m a n , et al.
(1981). Differences in sensitivity to temperature has also been established in this
work, as the temperature at which the hot water prevented potato was found to be
at 60oc for 25 minutes, whereas it varies in others. This has been ascribed to
natural variance between populations from different climates (D a s h w o o d et
al.,1991; H a r i k r i s h n a n and Y a n g , 2004). Dipping of ware potato tubers in
hot water at 60oC for 25 minutes in this humid tropical environment has been
found to be an effective means of reducing the spoilage of potato tubers during
storage without adversely affecting the quality of the tubers. This is in line with
what B u r n e t t et al. (1988) and R a n g a n n a , et al. (1998) discovered. However,
potato cultivars differ in sensitivity to hot water treatment, the temperature
detected in this work seems to be tolerated by these seed tubers especially in this
tropical environment, though for a limited period of time, without affecting their
sprouting and subsequent growth and yield. This method has also been discussed
by FAO, (1990) and L e a l a m and G a s h e , (1994) as one of the methods
approved to extend the shelf life of fresh potato tubers.
28
Abiodun Olusola Salami and Olumide Omololu Popoola
Conclusion
The control of Irish potato post harvest storage rot by thermal treatment have
proven to be very effective in checking post harvest storage rot in order to
prolong their shelf life in this study. This control method is culturally,
economically and socially suitable for the farmers. However, physical damage to
the root-tubers should be avoided at all cost since they serve as major entry
points for pathogens. Contact between the harvested root-tubers and the soil
should also be avoided, in order to prevent re-infection, since the soil is the
natural reservoir for pathogenic organisms. When in storage, root-tubers should
either be well spread out on wood or on clean concrete floor. The use of heated
soil which is free of pathogens could be considered as an alternative to water
where the latter is scarce.
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London, U.K. PP. 45-79.
Received, February 3, 2007
Accepted, July 16, 2007
TERMIČKA KONTROLA NEKIH UZROČNIKA TRULEŽI KRTOLA
KROMPIRA U SKLADIŠTU
Abiodun Olusola Salami1 and Olumide Omololu Popoola2
Rezime
U radu je ispitavan uticaj termičkog suzbijanja uzročnika truleži krtola
krompira u skaldištu. Ispitivane su tri lokalne sorte krompira: Patiska, Mai
Bowondoya i Nicola u Nigeriji. Iz obolelih krtola izolovene su sledeće
fitopatogene gljive: Botryodiploda theobromae, Fusarium redolens, Fusarium
oxysporum, Penicillium spp. i Rhizopus oryzae. Od tri ispitivane sorte krompira
Patiska je bila najotpornija na trulež, a zatim Mai Bawondoya, dok je Nicola biola
najosetljivija.
Pri ispitivanju uticaja izvora ugljenika na porast kolonija, utvrdjeno je da je
porast kolonija bio najveći na čorbi od krompira, a potom na čorbi od
Cocoyama i slatkog krompira, a najslabiji na čorbi od kasave. Potapanjem krtola
1
2
Ibadan
Abiodun Olusola Salami ,Plant Science Department, Obafemi Awolowo University, Ile-Ife
Abiodun Olusola Salami ,Botany And Microbiology Department, University Of Ibadan,
Thermal control of rot pathogens on potato
31
kromira u vodu zagrejanu na različitim temperaturama značajno je redukovana
populaciju patogena na njihovoj površini. Efikasnost tretiranja krtola toplom
vodom zagrejanom na 60°C je bila značajno viša nego pri teretiranju sa toplom
vodom zagrejanom na drugim temeparturama. Način tretiranja krtola krompira,
ispitivan u ovom radu je pokazao da patogeni uzročnici truleži uskladištenog
krompira (posebno kod seljaka) mogu biti uništeni bez smanjenja kvaliteta krtola
krompira.
Primljeno 3. februara 2007.
Odobreno16. jula 2007.