1. Introduction
Cowpea [Vignaunguiculata (L.) Walp] is an important legume in the midst of about 170 species of its genus because it’s an important source of protein and other essential nutrients for humans and animals [1]. Second African legume after groundnuts, its production faces many constraints such as insect attacks, drought, diseases... There are many diseases as cowpea brown blotch disease caused by Colletotrichumcapsici which is a very important disease of cowpea cultivation [Vignaunguiculata (L.) Walp] [2]. The typical symptoms characterizing it are observed at all stages of plant development, causing seed yield losses of the order of 75% [3]. Described as a disease subservient to the Sudanese and forested areas of cowpea cultivation in Africa [4], brown blotch disease is thought to be caused by a complex of [Colletotrichumcapsici (Syd.), Butler & Bisby] and [Colletotrichumtruncatum (Schein) Andrus & Moore] [5]. But authors such as Emechebe, [6]; Emechebe et al. [7] et Emechebe & Florini D. A. [8] established that Colletotrichumcapsici is the main pathogen of cowpea brown blotch disease because it contributes more than 90% in infections caused by it. Thus described as a disease of the Sudanese zones, the cowpea brown blotch disease is also encountered in the Sudano-Sahelian zone of Cameroon. This disease can cause wet losses in the field ranging from 42% to 100% [9].
However, the interest of this study is to highlight the diversity that would exist between Colletotrichumcapsici isolates, causal agent of the cowpea brown blotch disease in sudano-sahelian zone of Cameroon. Thus, following this introductive part, the structural organization of this work focuses on: Material and Methods: it was a question of presenting by describing the plant material used, the process of identification and isolation of isolates, the procedure of morphological and biometric characterization, and the data analysis, results and discussions presenting the proportions of isolates obtained by division, the morphological and biometric characteristics of the isolates, the diversity of isolates through their spatial arrangement in relation to the study parameters with a hierarchical ascending classification, a conclusion summarizing the results, a recommendation indicating the resulting research.
2. Materials and Methods
2.1. Plant Material
The plant material used in this study was constituted by organs (stems, leaves and pods) of cowpea plants with symptoms of cowpea brown blotch disease. They were collected in forty-four villages in seven divisions in the northern and far northern regions of the Sudano-Sahelian zone of Cameroon. The collection was done in the cowpea production basins according to the arrangement of the cardinal points [10]. These samples of diseased organs were transported to the plant pathology laboratory of the regional agricultural research center of Maroua.
2.2. Identification and Isolation of Colletotrichumcapsici Isolates
The infected organs collected were cut with scapel and then superficially desinfected with sodium hypochlorite (NaOCl) 1% for 5 minutes and immersed in 70% alcohol (v/v) for one minute followed by three successive rinses with sterile distilled water [11] [12]. After rinsing, the disinfected fragments were dried for at least two hours on sterile absorbent paper and then placed in the petri dishes previously dressed with three layers of blotting paper moistened with sterile distilled water. The boxes were incubated for 5 to 7 days at room temperature in the laboratory under alternating light and dark (12h/12h) [13]. After the incubation period, the cultured samples were taken under a stereo microscope for identification of Colletotrichumcapsici. Identification was made on the basis of the morphological characteristics of Colletotrichumcapsici using the identification key defined by Mathur and Kongsdal (2003) [14]. After identification, isolates were transplanted onto the Potato Dextrose Agar (PDA) culture medium supplanted by streptomycin and ampicilin and incubated for seven days for purification [15].
The single spores production was made from previously isolated and purified population isolates. A diameter of 5 mm conidian mass of each population isolate was transferred to 10 ml of sterile water. The mixture was stirred at the vortex and 1ml of the suspension was removed and added to 9 ml of sterile distilled water contained in a tube. The action was redone twice to obtain a less concentrated suspension (10−3 of the initial concentration) [16]. One to two drops of the suspension was sprayed on PDA medium. After sixteen to 24 hours of incubation, 3 to 4 sprouted conidia were individually transplanted onto PDA.
2.3. Morphological and Biometric Characterization of Single Spores Isolates of C. capsici
Morphological characterization was done based on qualitative and quantitative characteristics from the work of other scientists on different species of Colletotrichumspp. [17] [18] [19].
After purification of the single spore isolates, a 5 mm diameter fragment of each of the isolates was transferred and deposited in the centre of the petri dishes containing the PDA medium. The incubation of the cultures was done at room temperature of the laboratory (23˚C - 25˚C). The morphological characteristics selected for the study focused on the colony colour, the mycelial shape, the abundance of acervulis and the presence or absence of saltations [20]. Observations and notations were made on the seventh day after incubation of single spores isolates. The biometrics used for the study included mycelial diameter, daily radial growth rate, conidia length and width [21]. The reverses of petri dishes containing the single spores isolates were previously crossed by two diagonals (x, y) perpendicular to each other. The measurement of the evolution of the colony was taken every other day until the sixth day.
The mycelial diameter was calculated by the formula:
[18].
The radial growth rate was calculated by the formula:
[22]
where DMJn is the average growth diameter on the nth day of measurement.
The daily radial growth rate (DMJn) is the average daily increase in crop diameter; it’s calculated by the formula:
DMJn = (d1 + d2)/2 where d1 is the first diameter and d2 is the second diameter measured on the nth day.
DMJ0 = 5 mm = diameter of the transplanted explant.
On the tenth day after transplanting, 10 ml of sterile water was poured into the culture and after stirring, the suspension was returned to the test tubes. A drop was mounted on a micrometer and the reading was made using the Motic microscope at magnification 400. Thus, measurements (Length and width) of conidia were taken on 30 conidia per strain [23].
2.4. Data Analysis
The data was entered into the Excel 2010 spreadsheet. SPSS software was used for qualitative data analysis and STATGRAPHICS Centurion XVI.II software for quantitative data. These data were subjected to analysis of variance (ANOVA) and separate means using Fisher’s smallest significant difference test (PPDS) at the 5% probability threshold [24] for quantitative data and the khi two test for qualitative data. Using the averages of the measured and scored parameters, the principal component analysis was performed using XLSTAT 2007 software.
3. Results and Discussions
3.1. Results
The results obtained from this study are presented under labels such as identification of Colletotrichumcapsici isolates in Cameroon, their morphological characters, biometric characters, spatial arrangement and similarity.
3.1.1. Identification of Colletotrichumcapsici isolates in Cameroon
After collection, 151 (one hundred and fifty-one) samples of plant organs were collected. Of the samples collected, fifty-five (55) isolates of Colletotrichumcapsici were identified and isolated (Table 1). At least one isolate of C. capsici was identified in each division: 5.45% in Benoue, 7.27% in Mayo-Tsanaga, 10.90% in Mayo-Sava, 16.36% in Mayo-Louti, 14.55% in Mayo-Danay, 21.81% in Mayo-Kani and 23.64% in Diamare.
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Table 1. Single spores isolates of C. capsici identified in the Sudano-Sahelian zone of Cameroon.
Isolates were isolated from each type of organ collected in the field. Of the isolates identified, 50.92% are from the stem organ, 14.3% are isolated from the leaf organ and 34.55% are isolated from the pod organ.
3.1.2. Characteristics of Single Spores Isolates of C. capsici
1) Morphological characters
The results of the analysis of the colour data showed that there is a highly significant difference (P < 0.05) between isolates for colony colour. The colors found are: black, brown-black, gray, gray-pink, gray to black, gray to brown, brown to black, brown to brown and gray, brown-orange and gray, and brown to black (Figure 1). 1.81% of isolates are brown-orange and grey, and 36.36% of isolates are brown.
The results of the data analysis reveal that there is a highly significant difference (P < 0.05) between isolates for mycelial shape (Figure 2). Indeed, seven forms of mycelial are observed in C. capsici isolates: compact, compact to air, compact to cottony, aerial to compact, cottony to compact, cottony to air and aerial to cotton. However, the compact form is observed in 56.36% of isolates while the cotton to compact and aerial to cotton forms are observed in only 1.81% of C. capsici isolates.
The results of the data analysis showed that there is a highly significant difference (P < 0.05) between C. capsici isolates for acervulean abundance (Figure 3). Acervulis are absent in 1.81% of isolates, rare in 9.09% of isolates, scarce in 3.64%, very abundant in 12.7% of isolates, very abundant and well differentiated in 16.36% and abundant in 56.36% of C. capsici isolates.
The results of the analysis of the saltation data revealed that there is a highly
significant difference (P < 0.05) between C. capsici isolates in the presence or absence of saltations (Figure 4). Saltations are absent in 45.45% of isolates, present and poorly differentiated in 36.36% of isolates, present and well differentiated in 14.55% of isolates and, present and undifferentiated in 3.63% of C. capsici isolates.
2) Biometrics
The results of the analysis of mycelial diameter data showed that there is a statistically significant difference between isolates at 2 DAR (P < 0.05), 4 DAR (P < 0.05) and 6 DAR (P < 0.05). At 2 DAR, the mean mycelial diameter is 20.90 mm and isolate 089-MADI-153 has the smallest value (15.50 mm) in diameter mycelial while isolate 090-MADI-161 has the largest value (26.33 mm) in diameter mycelial. At 4 DAR, the average mycelial diameter is 44.23 mm. Isolate 089-MADI-153 has a smaller value (36.16 mm) and isolate 005-GUML-041 has the highest value (52.00 mm) in mycelial diameter. At 6 DAR, the average mycelial diameter is 66.23 mm and isolates 054-DIMD-071, 021-MEMS-061 and 048-SAMD-021 have the smallest (58.16 mm) and highest value (73.16 mm) in mycelial diameter, respectively.
In addition, the results of the analysis of the growth rate data showed that there is a statistically significant difference (P < 0.05) and the average growth rate is 11.03 mm/day. Isolate 005-GUML-041 has 6.69 mm/d (lowest value) and isolate 118-KAMK-212 has 12.33 mm/d. Multiple tests revealed 30 homogeneous groups. Table 2 presents the mycelial growth rate of C. capsisi isolates.
The results of the analysis of conidia length data showed that there is a statistically significant difference (P < 0.05) between C. capsici isolates for conidia length at 95% confidence. The conidia length of C. capsici varies between 48.10 ± 7.28 μm and 72.79 ± 1.05 μm with an average length of 64.31 ± 5.20 μm (Figure 5). Multiple scope tests matched 28 homogeneous groups.
The results of the analysis of conidia width data showed that there is a statistically significant difference (P < 0.05) between C. capsici isolates at 95% confidence. The width varies between 4.41 μm and 7.71 μm. However, the average width of the conidia is 5.30 ± 0.51 μm. Isolate 114-KOMK-173 has the smallest width value (4.41 ± 0.17 μm) and isolate 002-SOML-021 is the isolate with the
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Figure 4. Saltations of C. capsici isolates (%).
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Table 2. Mycelial growth rate (Mgr) of C. capsisi isolates.
highest width value (7.71 ± 1.86 μm) (Figure 6). Multiple scope tests matched 16 homogeneous groups.
Figure 6: Conidia width
The principal component analysis (PCA) made it possible to visualize that there are differences between the observed and measured characteristics: colony color (Cco), mycelial shape (Fmy), acervulean abundance (Aac), saltations (Sal), growth rate (Vcr), conidia length, conidia width (Figure 7). However, the representation of isolates studied following the evaluated traits shows that the distribution of these isolates is diversified in the Sudano-Sahelian zone. Thus, the isolates analyzed in the F1 × F2 biplot are visible at 47.15% and, yet 23.95% and 23.95% in the F1 and F2 axes respectively (Figure 8). Thus, there are ten (10) clouds grouping the isolates of said characters. Such a result shows that these traits are very important for discriminating against the C. capsici isolates studied. The traits used for the study were used by several scientists to characterize Colletotrichumspp. isolates, such as Smith and Black [25], Appiah-kubi et al. [26].
The first cloud shows that isolates 135-MABE-101, 138-NGBE-133, 139-NGBE-141, 042-MOMT-171, 009-SOML-083, 010-SOML-092, 015-SOML-143, 049-SAMD-032, 052-DIMD-062, 122-LAMK-253, 123-LAMK-263, 111-LAMK-193 are related by mycelial shape, saltations, colony colour and conidia width.
The 2nd cloud reveals that isolates 026-MAMT-011, 028-MAMT-031, 04-ZAMT-151, 017-TOMS-023, 021-TOMS-051, 013-GUML-123, 012-GUML-111, 011-GUML-101, 010-SOML-091, 047-SAMD-012, 057-TIMD-103, 049-SAMD-031, 048-SAMD021, 116-LAMK-191, 109-GUMK-121, 118-KAMK-212, 117-KAMK-203b, 097-GADI-231, 083-GRDI-091, 085-DODI-112b, 080-SADI-061, 090-MADI-151 and 094-GADI-201 are grouped around the abundance of acervulis, the rate of radial growth and conidia length.
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Cco: Color of the colony; Aac: Abundance of acervules; Sal: Saltations, Fmy: Mycelium form; Vcr: Radial growth rate, Lar spores: Spore width; Long spores: Spore length.
Figure 7. Spatial arrangement of the measured parameters.
The 3rd cloud presents isolates 017-TOMS-22, 017-TOMS-021, 021-MEMS-061, 107-TOMK-103, 085-DODI-111, 076-GUDI-023, 086-DODI-121 and 085-DODI-112a clustering around both conidia length and width.
The fourth, fifth and sixth clouds reveal only one isolate each; including isolates 019-TOMS-041, 002-SOML-021 and 005-GUML-041. Isolates 002-SOML-021 and 005-GUML-041 are closer to conidia width while isolate 019-TOMS-041 is closer to acervulis abundance.
The seventh cloud shows that isolates 054-DIMD-071, 051-SAMD-053, 089-MADI-151, 079-GUDI-053 and 089-MADI-153 cluster around the mycelial shape.
The eighth, ninth and tenth clouds each reveal a single isolate including 117-KAMK-203a, 114-KOMK-173 and 121-MOMK-243 respectively. Isolate 114-KOMK-173 is closer to the colony color and presence of saltations; 117-KAMK-203a isolate is close to the presence of saltations while 121-MOMK-243 isolate is close to mycelial form.
The hierarchical bottom-up classification (Figure 9) revealed the existence of ten (10) classes of isolates including several morphotypes of C. capsici, thus confirming the previous principal component analysis. Morphotypes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 comprise 23.64%, 41.82%, 14.55%, 1.82%, 1.82%, 1.82%, 9.09%, 1.82%, 1.82% and 1.82% of isolates, respectively.
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Figure 8. Spatial arrangement of isolates relative to parameters.
3.2. Discussions
After collection, 151 samples of plant organs were obtained. Of the 151 samples, 51 single spores isolates were isolated. These results show that isolates could not be identified and isolated at each collection site. However, Thio’s work (2016) [1] shows that at least one isolate has been identified in each of the collection sites.
From the same work, 44.44% of isolates were identified from the stem organ, 8.33% from the leaf organ and 47.22% from the pod organ compared to 50.92%, 14.3% and 34.55% respectively from the stem, leaf and pod organs in the present study. However, it should be noted that both in this study and in the other the high percentage of isolate comes from the stem organ, followed by the pod organ.
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Figure 9. Hierarchical bottom-up classification of Colletotrichum isolates.
This could be explained by the fact that Colletotrichumcapsici isolates come more from stems [25]. Also, the infectious process is highly developed during flowering and fruit formation [27].
The results of this study indicate that there are various colony colors of Colletotrichumcapsici: black, brown-black, brown-orange, brown, black, gray colors. These colors are characteristic of C. capsici and C. gloeosporiodes [28] [12].
This colour variation could be related either to the host plant or to the nature of the isolate or the environmental conditions of the isolate. The identification of Colletotrichum species is based on the morphocultural characteristics coupled with the origins of the isolates. However, several isolates of Colletotrichum show great variations in culture. [29].
Of the identified C. capsici isolates, 56.36% have abundant acervulis, which is much lower than the proportion (90%) observed by Thio (2018) [30]. In addition, Sereme [2] indicated that the abundance of acervulis is a discriminating character for the characterization of C. capsici.
Various forms of the mycelial have been noted such as compact, cottony, airy... These results corroborate those obtained by Ehui et al. [12].
The differences established between isolates by the different parameters confirm their use by several scientists including Ehui et al. [12]; Appiah et al. [26]; Sereme [2] characterized Colletotrichumspp isolates using these parameters. The revelation of several homogeneous groups of isolates indicates that there is a morpho-metric diversity of Colletotrichumcapsici in the Sudano-Sahelian zone of Cameroon.
The average growth diameter on the sixth day is 66.23 mm and smaller than that (68 mm) obtained by Sereme [2] and the average daily growth rate is 11.03 mm/day. These values are higher than those obtained by Ehui et al. [12] during the characterization of C. gloeosporiodes Penz manihotis causative agent of cassava anthracnose.
During his study, the average diameter was 65.40 mm and the radial growth rate was 9.20 mm/day. The average value of the growth diameter is higher than that obtained (57.87 mm) by Kuete et al. [17] when the radial growth rate is higher than that obtained by the same author (12.39 mm/day).
The conidia dimensions (length and width) observed in C. capsici isolates are not similar to those reported in other Colletrotrichum [18] [19], Appiah et al. [26] and similar to those reported in other Colletotrichum and fungal species Sereme [2] [12] [22].
Indeed, conidia lengths and widths ranged from 48.10 to 72.79 μm and 4.41 μm and 7.71 μm respectively. These values do not fall within the range described by Yun et al. [31]; who demonstrated that C. capsici conidia had dimensions ranging from 13.21 μm to 16.21 μm for length and from 1.79 to 3.28 μm for width.
4. Conclusion
Morphological and biometric variability could be preliminary for characterizing Colletotrichumcapsici. With diversity in morphological characters, conidial measurements, radial growth, mycelial colour in culture, presence or absence of saltations, it’s obvious that there are several morphotypes of isolates of Colletotrichumcapsici. Also, the similarities shown among some of the isolates from different divisions of sudano-sahelian zone of Cameroon suggest a common origin for some of them.
5. Recommendation
Following the results obtained from this study, it will be wise to evaluate the pathogenicity of this diversity of isolates and to conduct their Biomolecular caracterization.
Acknowledgements
The authors are thankful the Kirkhouse Trust Project for the material support provided for this work.