Abstract: Background and Objective: Early and late blight caused by Alternaria solani and Phytophthora infestans respectively, are the world’s most important diseases of tomato. The objective of the study was to assess the morphological and molecular diversity of A. solani and P. infestans in tomato growing farms in Kirinyaga, Kenya. Materials and Methods: Infected tomato leaf samples were obtained from tomato farms and cultured using V8 Agar for P. Infestans and PDA for A. solani to facilitate isolation of the pathogens. The isolates were then subjected to morphological characterization using microscopic and macroscopic features and molecular characterization through PCR amplification of their ITS regions. The PCR products were then sequenced and blasted using NCBI database. Results: The results showed high morphological and molecular diversity within A. solani but low genetic variability within P. infestans. At least four clones of A. solani were found to exist in the study area but only one strain of P. infestans was identified. Other disease-causing pathogens were also isolated from the samples including A. alternata, a fungus that causes leaf spot and other diseases in plants and Fusarium equiseti, a soil-borne fungus that causes wilt disease in different vegetable plants. Conclusion: These findings are useful in the development of sustainable strategies to manage the early and late blight and other related diseases in tomato growing areas in Kenya.
INTRODUCTION
Tomato (Solanum lycopersicum L.) is the second most important vegetable in Kenya after Brassicas1. In Kenya tomato contributes 14% of the entire vegetable produce and 7% of the entire horticultural produce2. The crop is cultivated for its fruits which are consumed by almost all the families to provide vitamins A, C and lycopene. Kenya is one of the major tomato producers in Sub-Saharan Africa (SSA) producing 410,033 tonnes of tomatoes annually3. Tomato also takes the lead in processed vegetable crops in Kenya4. However, tomato cultivation in Kenya is strained by a myriad of biotic and abiotic stresses, key among them being pests and diseases. Nowick et al.5 reported that over 200 pests and diseases attack the tomato plant thus hampering its production. Biotic agents of economic influence in tomato growing in Kenya include pests and diseases caused by bacteria, fungi, viruses and nematodes6.
Early and late blight caused by Alternaria solani and Phytophthora infestans respectively, are the most important diseases on tomato in the world6. Early blight is the most catastrophic disease-causing production and post-harvest losses resulting in 50-86% fruit yield cutback7. According to Desta and Yesuf8, high yield losses are recorded depending on disease severity. Torrential rain, high humidity and temperature range of 24-29°C creates a conducive environment for early blight development in tomatoes and can result to complete damage to the plant leaves9. Late blight is also a very disastrous tomato disease globally, causing remarkable economic losses annually5 especially under cool weather conditions10. In Kenya, the late blight together with the early blight was estimated to cause up to 95.8% of all the pre-harvest tomato losses11.
Alternaria solani is a soil-inhabiting fungus and spreads to host plants through air and rain splash9. The pathogen can survive for more than a decade in the soil, seed or crop remains at optimum temperature9. The pathogen produces toxins such as alternaric acid, altersolanol, macrosporin and zinniol that act on the protoplasm of the host and distract plant defence mechanisms12. Genetically, A. solani exhibits high variation between isolates from tomato and potato crops and different countries13. Isolates from unrelated host plant species vary in terms of aggressiveness, physiology and genetic diversity when inoculated in different plants13.
On the other hand, P. infestans is a diploid, obligate, biotrophic and heterothallic pathogen with two mating types: A1 and A214. The pathogen is an oomycete and its asexual lifecycle is distinguished by alternating phases of hyphal growth, sporangial germination and sporulation5. Sexual reproduction results in oospores that are thick-walled to enable them to overcome severe climatic conditions such as low temperatures, chemical fumigations and biodegradation, thus conserving the inocula for the subsequent years15. The pathogen has low levels of diversity and its population structure and distribution is influenced by host preference16.
Studying genetic variation within and among populations of related disease-causing agents is important in understanding pathogen-host co-evolution, disease epidemiology, development of sustainable control methods and preventing the development of host resistance17. For example, gene recombination through sexual reproduction can result in virulent genes thus complicating the management of diseases16. Genetic diversity of A. solani and P. infestans has been studied using various methods such as vegetative compatibility, virulence analyses, biochemical analyses and molecular analyses17. The most commonly used methods are molecular techniques such as isozyme analysis and PCR methods including Simple Sequence Repeats (SSR), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) markers13,18,19.
Different previous studies reported significant genetic variation within isolates of P. Infestans and A. solani. Van der Waals et al.17 observed an exorbitant level of genetic differences in A. solani using Random amplified microsatellite markers which were not typical of a species that is assumed to reproduce asexually only. Based on RAPD-profiles, Leiminger et al.20 reported the occurrence of significant genetic heterozygosity in A. solani isolates from potatoes in Southern Germany. Similar reports of the high degree of genetic diversity between isolates of A. solani were made by Nikam et al.21. Contrastingly, Cardenas et al.22 reported low genetic diversity among P. infestans isolates from crops within diverse Colombian regions and Venezuela. Similarly, Wu et al.s18 23 observed low genetic diversity among 134 strains of P. infestans from four provinces in China. Generally, minimal studies have been conducted on the genetic diversity of P. Infestans and A. solani in Kenya despite the significance of these pathogens in tomato and potato production. The current study targeted to examine the morphological and molecular diversity of A. solani and P. infestans isolated from tomato plants in farmers’ fields in Kirinyaga County, Kenya.
MATERIALS AND METHODS
Study area: The study was conducted between May to December, 2020. Sampling was done in Mwea, Kirinyaga County because it is an area renowned for tomato cultivation and blight diseases are rampant. The area lies within 0.6897°S, 37.3400°E. It is characterized by annual rainfall ranging between 800-1250 mm and is usually received in two seasons. The annual range of temperature is between 19.6-26.3°C. It has gentle rolling slopes with black cotton soils.
Collection of infected samples: Stratified random sampling was employed during the collection of samples whereby four tomato growing villages within the Sub-County were selected. Random sampling was done on fifteen tomato producing farms from each village for the collection of infected samples. Through visual examination, unhealthy tomato leaves showing symptoms of early and late blight were identified and randomly collected from the targeted farms. The collected diseased samples were put in cool boxes and transported to the University of Embu where they were preserved in a refrigerator at 4°C in the Microbiology Laboratory awaiting the pathogen isolation process.
Isolation of target pathogens: The isolation of P. Infestans and A. solani from the leaves that were infected was conducted following the modified approach of Naik et al.18 as adopted by Mugao et al.24. The tomato leaves bearing blight symptoms were washed under clean running tap water first before being surface sterilized in 1% sodium hypochlorite for three minutes. Rinsing was then done in three changes of sterilized distilled water and sterilized blotting paper was used to blot them dry. Infected leaf tissues of 3×3 mm size were cut using a sterilized scalpel towards the healthy tissues where the blight pathogens were suspected to be more active. Direct plating of the surface-sterilized tissues was done on the sterilized PDA and V8 agar for early and late blight independently and then incubated in the laboratory for three days at room temperature (25°C). Pure cultures were obtained through single spore isolation using a hyphal segment from the three-day-old colonies of each of the pathogens. The hyphal sections were introduced into a sterilized growth medium (PDA and V8 agar) and the incubation was done at room temperature.
Morphological characterization of the pathogen isolates: Identification of the pathogens was done 8 days after single spore isolation using morphological features based on established keys25 to verify the identity of the target pathogens. Morphological identification was based on visual observation of pathogen growth patterns, mycelia colours, margin colours and microscopic assessment of reproductive and vegetative structures25. Colonies with similar morphological characteristics were considered to be of the same species.
DNA extraction from pathogen isolates: The DNA extraction followed the modified procedure of Aamir et al.26. Sterilized wooden tooth sticks were used to aseptically scrap the mycelia of the 5-day old culture of A. solani and P. infestans isolates into separate Eppendorf tubes. In every tube, 1.5 mL Lysis buffer (50 mM Tris [pH 8.5], 20 mM EDTA [pH 8.0], 3% SDS) and 200 mg L–1 of 10 μL proteinase K were added and the content mixed by inversion. Vortexing of the mixture was done and then incubated at 65°C for 60 min. To the mixture, an equal volume of phenol-chloroform was added and the content was centrifuged at 13200 rpm for 5 min at 4°C. The supernatants were transferred to new tubes where 150 μL of sodium acetate (pH 5.2) was added and their volumes noted. An equal amount of isopropyl alcohol was added to the mixture and the content was mixed by inverting gently. The tubes were then spun at 13,200 rpm for 10 min and the supernatant discarded. To the resulting DNA pellets, 500 μL of 70% ethanol was added to wash them and then they were centrifuged for 1 min at 10,000 rpm and the supernatant was discarded. The DNA pellets were washed again with 70% ethanol and then re-suspended in 50 μL PCR water and stored at -20°C for further processing.
PCR amplification and DNA sequencing: PCR analysis was done in a 25 μL reaction mixture comprising 1 μL of genomic DNA, 0.5 μL each of the forward and reverse primers (ITS1 and ITS4), 0.125 μL of Taq polymerase, 2.5 mM dNTPs (2.5 μL), 0.125 μL of premix Taq buffer and 2.5 μL of 10x dream Taq buffer (MgCl2). The final volume was topped up with 17.75 μL of molecular grade water to make the 25 μL. Amplification was performed in a PCR with the following cycling conditions: an initial hybridization at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 2 min. The amplicons were resolved by gel electrophoresis in 1.2% agarose gel stained with ethidium bromide (0.5 μg mL–1). The DNA bands were visualized and photographed under UV light. The sizes of the amplicon were estimated by comparing them with a commercial 1 kb ladder RTU 1151021805 on the agarose gel. The primers ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCCTCC GCT TAT TGA TAT GC-3’) were used to amplify the Internal Transcribed Sequences (ITS) region. The PCR products were then sequenced and blasted using the NCBI database. Related species were identified based on more than 99% similarity between the query and reference sequence27,28.
RESULTS
Morphological identification of Alternaria isolates: Twenty-four Alternaria isolates suspected to be A. solani varied in their morphological characteristics. Conidia characteristics varied significantly between the isolates although they were all solitary. The observed morphological features characterized the isolates into five groups in Table 1. Group one was the largest comprising of twelve isolates that were characterized by an irregularly shaped colony of greenish-brown colour with dark brown substrate colour and greyish white margin in Fig. 1a-b. These isolates had septate conidia with four transverse septa and elongated but unbranched beak in Fig. 1c. Group two had four isolates characterized by a circular dark brown colony with dark grey substrate colour and brownish white margin in Fig. 2a-b. Members of this group had muriform septate conidia with three transverse septa, one longitudinal septum and an unbranched slender short beak in Fig. 2c. Group three comprised of two isolates with circular dark grey colony having a black substrate and a white margin in Fig. 3a and b. Their conidia were septate with three transverse septa, one longitudinal septum and elongated branched beak in Fig. 3c. Group four contained only one isolate (A23) with a circular colony of grey colour, brown zoned substrate and grey margin in Fig. 4a-b. The isolate had septate conidia with four transverse septa and an unbranched slender short beak in Fig. 4c. Group five consisted of five isolates with a circular greyish brown colony with a brownish white margin Fig. 5a-b. Their conidia were muriform with two transverse septa and one longitudinal septum and elongated branched beak in Fig. 5c. The mycelia for all the groups were septate, straight, hyaline and branched while the colour of the conidia was brown in the entire group.
Morphological identification of Phytophthora infestans: All the sixteen isolates suspected to be P. infestans did not vary in their morphological characteristics. The isolates had a white colony (front) colour and creamish substrate (reverse) colour. The growth pattern was circular with white margin colour in Fig. 6a-b. There were oospores with oogonia and amphigynous antheridia in Fig. 6c. The mycelia were aseptate, multinucleate and heterothallic in Fig. 6d.
Molecular identification of Alternaria solani: The PCR amplification of the ITS region of the twenty-four A. solani isolates resulted in a product of about 580 bp. The band size did not vary between the fungal isolates in Fig. 7. However, isolate A24 did not amplify and thus could not be positively identified through sequencing.
| Table 1: Morphological variability of Alternaria isolates | |||||||||
| Groups | Isolates | Colony color | Substrate color | Margin color | Margin growth | Transverse conidial septa | Longitudinal conidial septa | Beak elongation | Beak branching |
| 1 | A1, A2, A8, A10, A12, A13, A14, A15, A16, A18, A20, A22 | Greenish brown | Dark brown | Greyish white | Irregular | Four | None | Elongated | Unbranched |
| 2 | A4, A6, A9, A17 | Dark brown | Dark grey | Brownish white | Circular | Three | One | Slender short | Unbranched |
| 3 | A5, A11 | Dark grey | Black | White | Circular | Three | None | Elongated | Branched |
| 4 | A23 | Grey | Brown with zonation | Grey | Circular | Four | None | Slender short | Unbranched |
| 5 | A3, A7, A19, A21, A24 | Greyish brown | Greyish brown | Brownish white | Circular | Two | One | Stout short | Unbranched |
| Fig. 1(a-c): | Group one of the Alternaria isolates (a) Upper and (b) Lower and (c) Conidium |
| Fig. 2(a-c): | Group two of the Alternaria isolates (a) Upper, (b) Lower and (c) Conidium |
| Fig. 3(a-c): | Group three of the Alternaria isolates (a) Upper, (b) Lower and (c) Conidium |
| Fig. 4(a-c): | Group four of the Alternaria isolates (a) Upper, (b) Lower and (c) Conidium |
| Fig. 5(a-c): | Group five of the Alternaria isolates (a) Upper, (b) Lower and (c) Conidium |
| Fig. 6(a-d): | (a) Upper, (b) Lower colour of P. infestans colony, (c) Sporangiophore with oospores and (d) Mycelia |
Sequence similarity searches of the 23 isolates whose ITS fragment amplified successfully was carried out using the nucleotide BLAST program in the NCBI database. Nineteen of them were positively identified as A. solani while the other four were identified as A. alternata in Table 2. Eleven A. solani isolates showed 100% nucleotide similarity to Genebank clone 105 with accession number MN871613 together with isolate A1 which showed 99.42% nucleotide similarity to clone 105 in the Genebank. These had earlier been grouped under morphological group 1. Four other A. solani isolates (A4, A6, A9, A17) which were members of the morphological group 2 were found to be 100% similar to the Genebank clone 185 with accession number MN871616.
Isolates A5 and A11 which belonged to the morphological group 3 had 100% similarity with Genebank clone 43 with accession number MN871610 (Table 2). Isolate A23 which was the only one in morphological group 4 was found to have 99.81% nucleotide similarity with A. solani accession number LN879928 in the Genebank but its closest clone could not be identified. The accession MN871613 was the most prevalent with a total percent frequency of 52.18% followed by MN871616 (17.39%) and MN871610 (8.68%). The four isolates (A3, A7, A19, A21) that were identified to belong to Alternaria alternata resembled clones with accession numbers MN822496, MN822565, KY570321 and MW009021 respectively and they showed a 100% perfect match with different Genebank strains as shown in Table 2. The four belonged to morphological group 5 together with isolate 24 whose ITS fragment was not amplified.
| Fig. 7: | Gel image showing 580 bp (arrowed) DNA fragment amplified by ITS (1 and 4) primer in A. solani isolates on 1.2% agarose gel electrophoresis Ld is the I kb RTU 1151021805 ladder |
| Fig. 8: | Gel image showing 580 bp (arrowed) DNA fragment amplified by ITS (1 and 4) primer in P. infestans isolates on 1.2% agarose gel electrophoresis Ld is the I kb RTU 1151021805 ladder |
| Table 2: Molecular variability of Alternaria isolates | ||||||
| Groups | Isolate code | Species Identity |
Accession number |
Closest match |
Similarity (%) to Genebank accessions |
Frequency (%) |
| 1 | A1 | A. solani |
MN871613 |
Clone 105 |
99.42 |
4.35 |
| A2, A8, A10, A12, A13, A14, A15, A16, A18, A20, A22 | A. solani |
MN871613 |
Clone 105 |
100 |
47.83 |
|
| 2 | A4, A6, A9, A17 | A. solani |
MN871616 |
Clone 185 |
100 |
17.39 |
| 3 | A5, A11 | A. solani |
MN871610 |
Clone 43 |
100 |
8.68 |
| 4 | A23 | A. solani |
LN879928 |
Unidentified |
99.81 |
4.35 |
| 5 | A3 | A. alternata |
MN822496 |
BJ-YZ-14 |
100 |
4.35 |
| A7 | A. alternata |
MN822565 |
BJ-SB-41 |
100 |
4.35 |
|
| A19 | A. alternata |
KY570321 |
Strain Te 19 |
100 |
4.35 |
|
| A21 | A. alternata |
MW009021 |
DT1884-B |
100 |
4.35 |
|
Molecular identification of Phytophthora infestans: The ITS sequence amplification of the sixteen (16) P. infestans isolates by PCR resulted in a product of about 580 bp and there was no variation among the isolates in Fig. 8.
Sequence similarity searches of the 16 isolates suspected to be P. infestans was carried out using the nucleotide BLAST program which matched the isolates with Genebank accessions. The results showed that eleven of these isolates (68.75%) were positively identified as P. infestans with 100% genotypic similarity Strain A2 of accession number JX666330 in Table 3. Three other isolates (P8, P15, P16) belonged to unspecified species of the genus Phytophthora and matched 100% with Strain Phy-1i with Genebank accession number MT075724.
| Table 3: Molecular variability of Phytophthora | ||||||
| Groups | Isolate code | Species identity |
Accession number |
Closest match |
Similarity (%) to Genebank accessions |
Frequency (%) |
| 1 | P1, P2, P3, P4, P5, P7, P9, P10, P12, P13, P14 | P. infestans |
JX666330 |
Strain A2 |
100 |
68.75 |
| 2 | P8, P15, P16 | Phytophthora sp. |
MT075724 |
Strain Phy-1i |
100 |
18.75 |
| 3 | P6, P11 | Fusarium equiseti |
MK571264 |
Strain P3B |
100 |
12.5 |
The other two isolates (P6, P11) were identified as Fusarium equiseti with 100% resemblance to Strain P3B that had Genebank accession number MK571264.
DISCUSSION
The pathogenic, cultural, morphological and molecular characterization of pathogens has been attempted by many researchers in various countries18,21,29. In the current study, the pathogen isolates that were suspected to be twenty-four A. solani and sixteen P. infestans isolates until molecular identification revealed that they were mixed with other closely related fungal species. Therefore, morphological characterization provided a good lead towards species identification but could not specifically identify the isolates to species level. Okayo et al.30 noted that morphological classification of fungal species lacks precision but it is important in assisting the organization of the fungal isolates into groups allowing easier scrutiny by advanced methods.
Out of the 24 isolates that were initially thought to be A. solani, four of them were confirmed to belong to A. alternata, a fungus that causes leaf spot and other diseases in plants31. The two pathogens, A. alternata and A. solani, were reportedly isolated from blight infected plants in several previous studies. Zheng et al.32 reported the association of A. alternate and A. solani in causing potato blight in China. The two pathogens were also found to cause foliar diseases in Germany33. Loganathan et al.34 also reported A. alternata to cause early blight diseases in India with 80-90% disease incidence on susceptible tomato plants. However, A. alternata was absent in foliage with blight symptoms in Sweden35. On the other hand, out of the sixteen isolates that were suspected to be P. infestans, three of them belonged to unspecified species of the genus Phytophthora while two of them belonged to Fusarium equiseti which does a soil inhabit fungus known to cause wilt disease in different vegetable plants36.
Morphological characteristics such as colony colour, colony texture, size and shape of the conidia have been used to differentiate Alternaria species37 while asexual and sexual features are mostly used to differentiate Phytophthora species38. This study revealed high morphological variability within A. solani isolates but low morphological variation among P. infestans isolates. The macroscopic features such as growth pattern, colony (upper) colour, substrate (lower) colour and colour of the growth margin showed variability among A. solani isolates. Similar results were reported by Kumar et al.39, Tanvil et al.40, Brook and Dennis13 and Hubballi et al.41. In addition, microscopic features of mycelia and conidia of A. solani were variable but similar to those reported by Najibullah et al.42, Brooke and Dennis13 and Naik et al.18. Gannibal et al.43 also documented heterogeneity in various morphological attributes of A. solani.
High genetic diversity of A. solani have been reported by several authors20,44,45. Chaerani and Voorrips46 reported that genetic variation may occur among isolates obtained from different lesions of the same leaflet. According to Craven et al.47, genotypic variation in A. solani is caused by the ability of its mycelia to interconnect by bridges made through hyphal fusion that enable the distribution of nutrients, water and signalling molecules all over the colony. Genetic diversity is also contributed by mutations, selection, gene flow48, heterokaryosis which occurs as a result of hyphal anastomosis46, recombination and movement of the pathogen over long distances17.
The low genetic diversity observed in this study among P. infestans isolates corroborates the reports of Cardenas et al.22 and Wu et al.23 who also reported low diversity among isolates of P. infestans collected in the Northern Andean region and China respectively. Njoroge et al.49 also reported low variability within P. infestans in East Africa. The low variability among P. infestans isolates has been attributed to the existence of clonal populations of the pathogen in the target regions22-23. However, Han et al.50 reported high genetic diversity among P. infestans field isolates in China despite their high frequency of self-fertility. The possible sources of genetic variation in P. infestans include mitotic crossing over, gene conversion, extrachromosomal elements51, migration and sexual recombination of A1 and A2 mating types52-53. Other researchers attributed the diversity to self-sterility54, segregation of heterokaryons55, presence of many wild species of the host plant56 and ideal climate for the pathogen development57.
This study successfully identified A. solani and P. infestans isolates to species level through amplification and subsequent sequencing of their ITS region. The band size generated from the PCR product with ITS 1 and ITS 4 primers was about 580 bp for the two pathogens. These findings aligned with those of Loganathan et al.34 who used ITS1 and ITS4 to amplify the DNA of A. solani and obtained a band of 580 bp. Zheng et al.32 also obtained a 580 bp band in A. solani DNA amplified with primer set H3-1a/H3-1b. Reports of Manter and Vivanco58 and Embong et al.59 showed that different A. solani species generate bands ranging from 400-600 bp depending on the primers used. For P. infestans, different band sizes have been obtained by different researchers using different primers. The Genomic DNA of P. infestans amplified using TUBUF2 and TUBUR1 primers resulted in bands of about 990 bp20. Khalid et al.60 amplified the genomic DNA of P. infestans using ITS3 and ITS4 primers and yielded a band of 612 bp.
Sequencing and similarity matching of the isolates with available accessions through blasting on the NCBI database enabled identification of A. solani clones and P. infestans strains that are available in the study area. These findings indicated that different clones of A. solani existed in the study area which is not typical of a species that is known to reproduce only asexually. Clone 105 was the most prevalent with a total percent frequency of 52.18% followed by clone 185 (17.39%) and clone 43 (8.68%). The results also revealed the presence of one unidentified clone of A. solani in the study area. Van der Waals et al.17 and Leiminger et al.20 also reported the presence of different clones of A. solani in the same region in China and Southern Germany respectively. In addition, the results portrayed a close association between A. solani and A. alternata. The close association between the two pathogens in causing blight and other foliar diseases in plants have been reported by other researchers33-34. All the eleven isolates that were positively identified as P. infestans were found to be Strain A2 indicating high dominance of this strain in the study region. The high occurrence of the A2 strain of P. infestans has been reported in several parts of China50,61,62.
CONCLUSION
This study concluded that there is higher genetic variability within A. solani than P. infestans in Kirinyaga County, Kenya. At least four clones of A. solani were identified in the area including clone 105 which was the most dominant, clone 185 and clone 43. However, only one strain of P. infestans (Strain A2) was identified in the study region. The study also revealed a close association between A. solani and A. alternata as well as between P. infestans and other identified Phytophthora species. The variability of the pathogens can be studied across seasons and different host plants to understand the changes in epidemiology and host-pathogen interactions. It may also be important to investigate the nature of the association between the target pathogens and the other pathogens identified in the area. In addition, the study revealed the presence of Fusarium equiseti a soil-borne fungus that causes wilt disease in different vegetable plants.
SIGNIFICANCE STATEMENT
This study discovered that the early blight pathogen A. solani exist in more variable forms than the late blight pathogen P. infestans in Kirinyaga County, Kenya. These findings will be useful in the development of sustainable strategies to manage the early and late blight diseases in tomato growing areas in Kenya. The close association revealed by this study between A. solani and A. alternata as well as between P. infestans and another unidentified phytophthora species will form the basis of further research to determine the nature of that association.
ACKNOWLEDGMENT
The authors acknowledge the Higher Education Loans Board for providing financial support and the University of Embu for providing the laboratory space where the work was carried out.