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Studies on the post-harvest rots of African breadfruit (Treculia africana) seeds in Nigeria
International Biodeterioration 24 (1988) 17-23
Studies on the Post-harvest Rots of African Breadfruit (Treculia africana) Seeds in Nigeria
M. I. N w u f o & P. C. M b a School of Agriculture and Agricultural Technology, Federal University of Technology, Owerri, Nigeria (Received 10 August 1987; accepted 17 September 1987)
ABSTRACT A survey of the storage rot diseases ofTreculia a fricana was conducted in Imo State, Nigeria in 1985-86. The storage loss was about 15.2% during severe infection. Four types of microorganisms, namely Botryodiplodia theobromae, Rhizopus stolonifer, Aspergillus niger and yeasts, were consistently isolated from the rotten seeds. A~ niger, B. theobromae and R. stolonifer were associated with about 90% of the total loss. This is the first report of the occurrence of these microorganisms associated with T. africana seeds in Nigeria. The ability of each to cause storage rot of seeds was established and the effect of temperature, p H and different carbon sources on growth of the organisms investigated.
INTRODUCTION African breadfruit (Treculia africana), a m e m b e r of Moraceae, is a fruit tree which grows uncultivated a n d semi-wild. It produces mature fruits during the ' h u n g r y season' in I m o a n d A n a m b r a States, Nigeria, following the planting of m a j o r staples such as yams, maize a n d cocoyam. T h e seeds contain about 17-23% crude protein a n d are cooked a n d eaten as a m a i n dish. A n a z o n w u (1981) reported that the fruits ofT. afn'cana can be processed into breadfruit flour from which several food products such as bread, cookies a n d biscuits can be prepared. The seeds 17 International Biodeterioration 0265-3036/88/$03.50©1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain.
18
M. I. Nwufo & P C. Mba
are not stored for long periods because they are highly perishable. M u c h work has been d o n e on the storage diseases of m a n y fruits, roots a n d tubers in Nigeria but no c o m p r e h e n s i v e investigation has been carried out o n the storage rot diseases of the seeds of T. africana in Nigeria. T. africana is one of the neglected tree crops in Nigeria a n d literature is scanty on the origin, distribution a n d a g r o n o m y of the crop. It is on this premise that a survey of the storage diseases ofT. africana was c o n d u c t e d in I m o State, Nigeria. The effects of e n v i r o n m e n t a l factors such as temperature, p H a n d different c a r b o n sources on the associated organisms are presented in this paper.
MATERIALS AND METHODS A survey of the storage diseases o f the seeds ofT. africana was carried out in 1985 a n d 1986 in five locations in I m o State. The locations included Owerri, U m u a h i a , Etiti, Aba a n d Arochukwu. The incidence of the seed rots was assessed as a percentage o f the total n u m b e r of 10 000 seeds in each location. Isolations were carried out by removing small portions (about 2 m m ) of the diseased seeds a n d plating o n potato dextrose agar (PDA) after surface sterilization with 3% solution of calcium hypochlorite a n d removal of the testa of the seeds. To test for the capacity to cause storage rot, pure isolates from the infected seeds were inoculated into healthy seeds. The testa of the seeds was r e m o v e d a n d the seeds were washed, surface-sterilized a n d rinsed in sterile distilled water. All inoculated seeds were incubated u n d e r high relative h u m i d i t y at 25°C for the first 48 h a n d later transferred into incubators for 7 days. The seeds were e x a m i n e d for rot development. The effect o f different c a r b o n sources on the growth a n d sporulation o f the fungi was d e t e r m i n e d using a carbon-free sterilized basal m e d i u m containing NI-I4NO3,1.0 g; KH2PO4, 1.5 g; MgSO4.7H20, (} 5 g; CaC12.2H20, 0-1 g; NaC1, 0.01 g a n d 2 m l of micronutrient stock solution. The micronutrient solution c o n t a i n e d 1.2 g FeC13.6H20; 1.6 g ZnSO4.7H20; 0.4 g each of COC12.6H20, KI a n d H3BO3 a n d 0.20 g each of CuSO4 a n d MnC12.4H20 in 100 ml of distilled water. A 1% (w/v) solution of glucose, sucrose, fructose, maltose, starch or dextrin was incorporated into the m e d i u m at the rate of 10 ml to 20 ml basal m e d i u m . Flasks o f the liquid m e d i u m were each inoculated with a 5 - m m mycelial disc of the isolated fungi a n d incubated at 30°C for 7 days w h e n dry weight determinations were m a d e (see below). The effects of the variation of temperature a n d p H on the growth of the
Post-harvest rots ofT. africana seeds
19
rot p a t h o g e n s were investigated. F o r the effect of temperature on the growth of the fungi flasks c o n t a i n i n g 30 ml sterile m e d i u m (potato dextrose m e d i u m ) were inoculated with a disc (5 m m diameter) o f fungal culture obtained from the m a r g i n of actively growing colonies of each o r g a n i s m a n d placed in incubators m a i n t a i n e d at 5, 10, 20, 25, 30, 35 a n d 40°C. Ten flasks were used for each isolate at each temperature. Mycelial harvesting was d o n e by filtering with pre-weighed oven-dried filter papers ( W h a t m a n N o 1). All filtered mycelia were dried in an oven at 75°C to constant weight. Variations o f p H (2-9) were obtained by adjusting the p H of the media with citrate or borate buffer. Mcllvaine's citrate buffer solution was used to obtain p H 9. T h e adjustments were m a d e aseptically after sterilization. The effect o f p H on the growth of the different isolates was investigated by inoculating flasks of liquid m e d i u m adjusted to the required p H levels (2-9) a n d incubating at 30°C for 7 days w h e n the growth determinations were made.
RESULTS D u r i n g the survey it was observed that the m e t h o d of processing the seeds from the fruits predisposed t h e m to m e c h a n i c a l damage. The w o u n d s served as points of entry for the fungi. The incidence of storage rots was high in all the locations (Table 1) except Arochukwu. Table 2 s u m m a r i z e s the microorganisms isolated from diseased seeds of T. africana. Results showed that the following fungi: Aspergillus niger, TABLE 1 The Incidence of Storage Rots of Treculia afn'cana Determined at the Different Locations Location
Incidence (% +_SE)
Owerii
10'5 _+ 1-5
Umuahia
15.2 + 0.3
Etiti
8"5 _+0-6
Aba
6"3 + 0-3
Arochukwu
2"3 _+0' 1
Data are means of 3 determinations from the different locations.
M. L Nwufo & P C Mba
20
TABLE 2 The Percentage of Rotted Seeds Yielding Different Fungi in Different Locations of Imo State
% Rot at different locations
Pathogen
A. niger 17. stolonifer B. theobromae Yeasts
Owerri
Umuahia
Etiti
Aba
39"6 + 1"2 47'5 + 0"8 22"4 + 1"2 11"2+0-4
40"6+ 1"6 59"4 + 2'1 13'6 + if4 __a
47"5 + 1"3 34-2 + 6"8 21-6 +_ 0"2 10"8+0"1
Arochukwu
44-2 +__0"8 53"6 _+ 4'6 11-8 +_ 2-2 9"5+__0'1
41.5 _+ 6"2 51"2 +_ 6-1 20-8 + I)4 --a
aNot isolated from the location. Percentage for each rot is expressed as percentage of total rots.
Rhizopus stolonifer, Botryodiplodia theobromae and yeasts were the predominant fungi assocated with the biodeterioration of T. africana in Imo State. Rot in seeds inoculated with Botryodiplodia was characterized by water-soaked lesions which spread gradually. Lesions started as a ___x x----x ; o o /X /k
A niger Yeast B theobromae R stolonifer
\ \
E o03 E~
E
\
v
.~_~100 >., ! ! J
0
5
10
15 20 25 30 T e m p e r a t u r e (°C)
35
40
Fig. 1. Effect of temperature on the growth of the four rot pathogens in liquid potato dextrose medium.
Post-harvest rots of T. africana seeds
21
greyish colour which eventually turned black. The Rhizopus rot was also characterized by water-soaked lesions. The rot spread rapidly and the seeds completely rotted after 3 days of incubation. The whitish mycelium of the fungus grew on the surface of the seeds. Aspergillus rot was characterized by the development of dry, firm decaying areas. The seeds were discoloured to purple-brown. Yeast rot was characterized by the softening of the seed and the production of watery fluid usually accompanied by pungent odour. Results of experiments to show the effect of temperature on the growth of the four isolates (Fig. 1) showed that all the microorganisms grew at temperatures above 10°C. The optimum temperature range for the growth of the four organisms was found to be 30-35°C. Only B. theobromae did not grow at 40°C. The optimum pH for the growth of the four microorganisms was pH 34. All the organisms sporulated between pH 2 and pH 10 (Fig. 2). The results of the effect of different carbon sources on the growth of the organisms showed that the five carbon sources supported the growth of the organisms to varying degrees. Starch was found to be the best carbon
x o = A
200
× o : Z~
A niger B theobromae Yeast R stolonifer
~5 @ E 0
E ~ IO0
} 5o
o
2
~
4
6
pH
values
$
7
8
9
Fig. 2. Effect of p H on the growth of the four rot pathogens in liquid potato dextrose medium.
22
M. I. Nwufo & P. C. Mba
~ Fructose 200
~
~:~Maltose
m×~ NSucrose E E 15o
l
/
~xx
~
Glucose
~
~
Starch
x~
~g
ot ~
/
×' X X XX
E
77
,','
.
,
, ",,', ,',"
2/
:::4
//
:::q
~)
,
x x
//
::: ,× ×
/,-
'..-,
[
xA
:,5:
//
•
/×
, ,x~l "',,~
• ,
xx]
!:):t
,'.s \x,,
"d,"
B theobromae
,,
,'.'- )K)q //
~_ 50
, \
Yeast Aspergill us Pathogens
R stolonifer
Fig. 3. Utilization of different carbon sources by the rot pathogens at 30°C.
source for the growth ofB. theobromae, A. niger and yeast., while maltose was the best carbon source for the growth of R. stolonifer (Fig. 3).
DISCUSSION This first report of the assessment of the storage losses of African breadfruit seeds in Nigeria shows a high incidence of post-harvest rots (2.3-15.2%) resulting in high storage losses of the seeds of T. africana in Imo State. Four types of fungi, Aspergillus niger, Rhizopus stolonifer, Botryodiplodia theobromae and yeasts were implicated as rot pathogens of T. africana. None of these pathogens has been reported in any part of the world on the seeds of T. africana. A. niger and R. stolonifer were found to be the major causal organisms involved in the storage rots of the crop in Imo State, Nigeria. Results showed that these isolates were capable of causing disease if the right conditions are available. The fact that B. theobromae grew at 10°C but not at 40°C is broadly in line with the findings of Goos et al. (1961) that the minimum, optimum and maximum temperatures for the growth of an isolate ofB. theobromae
Post-harvest rots ofT. africana seeds
23
from Musa spp. were 15, 25-30 and 37°C, respectively. However, O g u n d a n a et al. (1971) reported that the m i n i m u m , optimum and m a x i m u m for an isolate ofB. theobromae from yam (Dioscorea) were 10, 26-30 and 40°C. Nwufo (1980) also reported that B. theobromae isolated from cocoyam (Colocasia) had m i n i m u m , optimum and m a x i m u m temperatures for growth at 15, 25-30 and 35°C. These differences in the results obtained by different workers are slight and can probably be explained in terms of physiological differences between strains. Investigations are in progress on the effects of environmental conditions on disease development, biochemical changes in infected seeds and control of the seed rot diseases of T. africana, an important staple food crop in Imo State.
REFERENCES Anazonwu, J. N. (1981). Indigenous food and nutritional adequacy. Symposium on the Development of Indigenous Technology, Ministry of Science and Technology, Enugu, 29-30 October, 1981. Goos, R. D.; Cox, E. A.; Siotzky, P. (1961). Botryodiplodia theobromae and its association with Musa sp. Mycologia 53, 262-77. Nwufo, M. I. (1980). Studies of storage rot diseases of cocoyam (Colocasia esculenta). M.Sc. Dissertation, University of Ibadan. Ogundana, S. K~;Naqvi, S. H.; Ekundayo, J. A. (1971). Studies of soft rot of yams in storage Transactions of the British Mycological Society 56, 73-80.
Studies on the Post-harvest Rots of African Breadfruit (Treculia africana) Seeds in Nigeria
M. I. N w u f o & P. C. M b a School of Agriculture and Agricultural Technology, Federal University of Technology, Owerri, Nigeria (Received 10 August 1987; accepted 17 September 1987)
ABSTRACT A survey of the storage rot diseases ofTreculia a fricana was conducted in Imo State, Nigeria in 1985-86. The storage loss was about 15.2% during severe infection. Four types of microorganisms, namely Botryodiplodia theobromae, Rhizopus stolonifer, Aspergillus niger and yeasts, were consistently isolated from the rotten seeds. A~ niger, B. theobromae and R. stolonifer were associated with about 90% of the total loss. This is the first report of the occurrence of these microorganisms associated with T. africana seeds in Nigeria. The ability of each to cause storage rot of seeds was established and the effect of temperature, p H and different carbon sources on growth of the organisms investigated.
INTRODUCTION African breadfruit (Treculia africana), a m e m b e r of Moraceae, is a fruit tree which grows uncultivated a n d semi-wild. It produces mature fruits during the ' h u n g r y season' in I m o a n d A n a m b r a States, Nigeria, following the planting of m a j o r staples such as yams, maize a n d cocoyam. T h e seeds contain about 17-23% crude protein a n d are cooked a n d eaten as a m a i n dish. A n a z o n w u (1981) reported that the fruits ofT. afn'cana can be processed into breadfruit flour from which several food products such as bread, cookies a n d biscuits can be prepared. The seeds 17 International Biodeterioration 0265-3036/88/$03.50©1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain.
18
M. I. Nwufo & P C. Mba
are not stored for long periods because they are highly perishable. M u c h work has been d o n e on the storage diseases of m a n y fruits, roots a n d tubers in Nigeria but no c o m p r e h e n s i v e investigation has been carried out o n the storage rot diseases of the seeds of T. africana in Nigeria. T. africana is one of the neglected tree crops in Nigeria a n d literature is scanty on the origin, distribution a n d a g r o n o m y of the crop. It is on this premise that a survey of the storage diseases ofT. africana was c o n d u c t e d in I m o State, Nigeria. The effects of e n v i r o n m e n t a l factors such as temperature, p H a n d different c a r b o n sources on the associated organisms are presented in this paper.
MATERIALS AND METHODS A survey of the storage diseases o f the seeds ofT. africana was carried out in 1985 a n d 1986 in five locations in I m o State. The locations included Owerri, U m u a h i a , Etiti, Aba a n d Arochukwu. The incidence of the seed rots was assessed as a percentage o f the total n u m b e r of 10 000 seeds in each location. Isolations were carried out by removing small portions (about 2 m m ) of the diseased seeds a n d plating o n potato dextrose agar (PDA) after surface sterilization with 3% solution of calcium hypochlorite a n d removal of the testa of the seeds. To test for the capacity to cause storage rot, pure isolates from the infected seeds were inoculated into healthy seeds. The testa of the seeds was r e m o v e d a n d the seeds were washed, surface-sterilized a n d rinsed in sterile distilled water. All inoculated seeds were incubated u n d e r high relative h u m i d i t y at 25°C for the first 48 h a n d later transferred into incubators for 7 days. The seeds were e x a m i n e d for rot development. The effect o f different c a r b o n sources on the growth a n d sporulation o f the fungi was d e t e r m i n e d using a carbon-free sterilized basal m e d i u m containing NI-I4NO3,1.0 g; KH2PO4, 1.5 g; MgSO4.7H20, (} 5 g; CaC12.2H20, 0-1 g; NaC1, 0.01 g a n d 2 m l of micronutrient stock solution. The micronutrient solution c o n t a i n e d 1.2 g FeC13.6H20; 1.6 g ZnSO4.7H20; 0.4 g each of COC12.6H20, KI a n d H3BO3 a n d 0.20 g each of CuSO4 a n d MnC12.4H20 in 100 ml of distilled water. A 1% (w/v) solution of glucose, sucrose, fructose, maltose, starch or dextrin was incorporated into the m e d i u m at the rate of 10 ml to 20 ml basal m e d i u m . Flasks o f the liquid m e d i u m were each inoculated with a 5 - m m mycelial disc of the isolated fungi a n d incubated at 30°C for 7 days w h e n dry weight determinations were m a d e (see below). The effects of the variation of temperature a n d p H on the growth of the
Post-harvest rots ofT. africana seeds
19
rot p a t h o g e n s were investigated. F o r the effect of temperature on the growth of the fungi flasks c o n t a i n i n g 30 ml sterile m e d i u m (potato dextrose m e d i u m ) were inoculated with a disc (5 m m diameter) o f fungal culture obtained from the m a r g i n of actively growing colonies of each o r g a n i s m a n d placed in incubators m a i n t a i n e d at 5, 10, 20, 25, 30, 35 a n d 40°C. Ten flasks were used for each isolate at each temperature. Mycelial harvesting was d o n e by filtering with pre-weighed oven-dried filter papers ( W h a t m a n N o 1). All filtered mycelia were dried in an oven at 75°C to constant weight. Variations o f p H (2-9) were obtained by adjusting the p H of the media with citrate or borate buffer. Mcllvaine's citrate buffer solution was used to obtain p H 9. T h e adjustments were m a d e aseptically after sterilization. The effect o f p H on the growth of the different isolates was investigated by inoculating flasks of liquid m e d i u m adjusted to the required p H levels (2-9) a n d incubating at 30°C for 7 days w h e n the growth determinations were made.
RESULTS D u r i n g the survey it was observed that the m e t h o d of processing the seeds from the fruits predisposed t h e m to m e c h a n i c a l damage. The w o u n d s served as points of entry for the fungi. The incidence of storage rots was high in all the locations (Table 1) except Arochukwu. Table 2 s u m m a r i z e s the microorganisms isolated from diseased seeds of T. africana. Results showed that the following fungi: Aspergillus niger, TABLE 1 The Incidence of Storage Rots of Treculia afn'cana Determined at the Different Locations Location
Incidence (% +_SE)
Owerii
10'5 _+ 1-5
Umuahia
15.2 + 0.3
Etiti
8"5 _+0-6
Aba
6"3 + 0-3
Arochukwu
2"3 _+0' 1
Data are means of 3 determinations from the different locations.
M. L Nwufo & P C Mba
20
TABLE 2 The Percentage of Rotted Seeds Yielding Different Fungi in Different Locations of Imo State
% Rot at different locations
Pathogen
A. niger 17. stolonifer B. theobromae Yeasts
Owerri
Umuahia
Etiti
Aba
39"6 + 1"2 47'5 + 0"8 22"4 + 1"2 11"2+0-4
40"6+ 1"6 59"4 + 2'1 13'6 + if4 __a
47"5 + 1"3 34-2 + 6"8 21-6 +_ 0"2 10"8+0"1
Arochukwu
44-2 +__0"8 53"6 _+ 4'6 11-8 +_ 2-2 9"5+__0'1
41.5 _+ 6"2 51"2 +_ 6-1 20-8 + I)4 --a
aNot isolated from the location. Percentage for each rot is expressed as percentage of total rots.
Rhizopus stolonifer, Botryodiplodia theobromae and yeasts were the predominant fungi assocated with the biodeterioration of T. africana in Imo State. Rot in seeds inoculated with Botryodiplodia was characterized by water-soaked lesions which spread gradually. Lesions started as a ___x x----x ; o o /X /k
A niger Yeast B theobromae R stolonifer
\ \
E o03 E~
E
\
v
.~_~100 >., ! ! J
0
5
10
15 20 25 30 T e m p e r a t u r e (°C)
35
40
Fig. 1. Effect of temperature on the growth of the four rot pathogens in liquid potato dextrose medium.
Post-harvest rots of T. africana seeds
21
greyish colour which eventually turned black. The Rhizopus rot was also characterized by water-soaked lesions. The rot spread rapidly and the seeds completely rotted after 3 days of incubation. The whitish mycelium of the fungus grew on the surface of the seeds. Aspergillus rot was characterized by the development of dry, firm decaying areas. The seeds were discoloured to purple-brown. Yeast rot was characterized by the softening of the seed and the production of watery fluid usually accompanied by pungent odour. Results of experiments to show the effect of temperature on the growth of the four isolates (Fig. 1) showed that all the microorganisms grew at temperatures above 10°C. The optimum temperature range for the growth of the four organisms was found to be 30-35°C. Only B. theobromae did not grow at 40°C. The optimum pH for the growth of the four microorganisms was pH 34. All the organisms sporulated between pH 2 and pH 10 (Fig. 2). The results of the effect of different carbon sources on the growth of the organisms showed that the five carbon sources supported the growth of the organisms to varying degrees. Starch was found to be the best carbon
x o = A
200
× o : Z~
A niger B theobromae Yeast R stolonifer
~5 @ E 0
E ~ IO0
} 5o
o
2
~
4
6
pH
values
$
7
8
9
Fig. 2. Effect of p H on the growth of the four rot pathogens in liquid potato dextrose medium.
22
M. I. Nwufo & P. C. Mba
~ Fructose 200
~
~:~Maltose
m×~ NSucrose E E 15o
l
/
~xx
~
Glucose
~
~
Starch
x~
~g
ot ~
/
×' X X XX
E
77
,','
.
,
, ",,', ,',"
2/
:::4
//
:::q
~)
,
x x
//
::: ,× ×
/,-
'..-,
[
xA
:,5:
//
•
/×
, ,x~l "',,~
• ,
xx]
!:):t
,'.s \x,,
"d,"
B theobromae
,,
,'.'- )K)q //
~_ 50
, \
Yeast Aspergill us Pathogens
R stolonifer
Fig. 3. Utilization of different carbon sources by the rot pathogens at 30°C.
source for the growth ofB. theobromae, A. niger and yeast., while maltose was the best carbon source for the growth of R. stolonifer (Fig. 3).
DISCUSSION This first report of the assessment of the storage losses of African breadfruit seeds in Nigeria shows a high incidence of post-harvest rots (2.3-15.2%) resulting in high storage losses of the seeds of T. africana in Imo State. Four types of fungi, Aspergillus niger, Rhizopus stolonifer, Botryodiplodia theobromae and yeasts were implicated as rot pathogens of T. africana. None of these pathogens has been reported in any part of the world on the seeds of T. africana. A. niger and R. stolonifer were found to be the major causal organisms involved in the storage rots of the crop in Imo State, Nigeria. Results showed that these isolates were capable of causing disease if the right conditions are available. The fact that B. theobromae grew at 10°C but not at 40°C is broadly in line with the findings of Goos et al. (1961) that the minimum, optimum and maximum temperatures for the growth of an isolate ofB. theobromae
Post-harvest rots ofT. africana seeds
23
from Musa spp. were 15, 25-30 and 37°C, respectively. However, O g u n d a n a et al. (1971) reported that the m i n i m u m , optimum and m a x i m u m for an isolate ofB. theobromae from yam (Dioscorea) were 10, 26-30 and 40°C. Nwufo (1980) also reported that B. theobromae isolated from cocoyam (Colocasia) had m i n i m u m , optimum and m a x i m u m temperatures for growth at 15, 25-30 and 35°C. These differences in the results obtained by different workers are slight and can probably be explained in terms of physiological differences between strains. Investigations are in progress on the effects of environmental conditions on disease development, biochemical changes in infected seeds and control of the seed rot diseases of T. africana, an important staple food crop in Imo State.
REFERENCES Anazonwu, J. N. (1981). Indigenous food and nutritional adequacy. Symposium on the Development of Indigenous Technology, Ministry of Science and Technology, Enugu, 29-30 October, 1981. Goos, R. D.; Cox, E. A.; Siotzky, P. (1961). Botryodiplodia theobromae and its association with Musa sp. Mycologia 53, 262-77. Nwufo, M. I. (1980). Studies of storage rot diseases of cocoyam (Colocasia esculenta). M.Sc. Dissertation, University of Ibadan. Ogundana, S. K~;Naqvi, S. H.; Ekundayo, J. A. (1971). Studies of soft rot of yams in storage Transactions of the British Mycological Society 56, 73-80.