Assessment of fungal pathogens of cabbage (Brassica oleracea L. brassicaceae) in the western highlands of Cameroon (A BSPP ‘Lockdown’ Bursary Report)

This is the report from a BSPP Lockdown Bursary.
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Today, one of the main global challenges is how to ensure food security for a world growing population whilst ensuring long-term sustainable development. Fungal pathogens of cabbage contribute significantly to both pre-harvest and post-harvest losses leading to food insecurity which affect the nutritional status and health of populations. According to the FAO, food production will need to grow by 70% to feed world population which will reach 9 billion by 2050.

Recent approaches to agricultural development, including food production and food security, have largely failed to reduce the absolute numbers of the food insecure or to ensure environmental sustainability. Whilst global achievements in food production have been impressive in the last 50 years, the global inequalities in food entitlements (that is people’s ability to acquire food and gain access to and control of productive resources) remain one of the biggest obstacles to achieving food security for all. Many assume that increasing food production is the only necessary condition for improving food security to achieve the millennium development goal. It is clearly important, especially in the complex, diverse, risk-prone agriculture. Cabbage (Brassica oleracea L.) is one of the most important vegetables grown worldwide. It belongs to the family Brassicaceae or Cruciferae, which includes broccoli, cauliflower, and kale. The different cultivated types of cabbage show great variation in respect of size, shape and color of leaves as well as the texture of the head. Approximately 6.3 kg of Brassica vegetables are consumed per person annually. Great producers of this vegetable crops in the world are China, India and the United States with China being the largest producer.

Generally, cabbages (green and red) contain different patterns of glucosinolates specially singrin which received special attention in cancer prevention, also the rich red color of some cabbage cultivars reflects its concentration of anthocyanin polyphenols which considered a strong antioxidant having a therapeutic role in a number of diseases. In addition to fiber related components, cabbages contain different vitamins such as vitamin A and C, minerals, sugars, carbohydrates and proteins that increase its nutritional value. Cabbage can be affected by diseases in all stages of growth, thus typical symptoms that appear on the cabbage leaves include black necrotic lesions surrounded by chlorotic areas on leaves, stems and siliquae, appear. Fungal diseases result to losses in both quality and quantity. Quality losses include those that affect the nutrient/caloric composition, the acceptability, and the edibility of a given product. These losses are generally more common in developed countries. Quantity losses refer to those that result in the loss of the amount of a product. Loss of quantity is more common in developing countries. A recent FAO report indicates that at global level, volumes of lost and wasted food in high income regions are higher in downstream phases of the food chain, but just the opposite in low-income regions where more food is lost and wasted in upstream phases.

A good number of fungal diseases affect cabbage production in the field which include alternaria leaf spot, fusarium, penicillium, downy mildew, phytophthora root rot, powdery mildew, ring spot, white mold result in the partial or total destruction of the whole plant. As a results of the devastating effects of the fungal diseases on the farmers and the economy, a good number of strategies have been put in place to reduce the diseases to a manageable level in other to protect the health of consumers. However, only baseline research have been conducted in the regions (Santa and Dschang) of Cameroon on fungal diseases of cabbage.

The first objective of this study was to determine the incidence, severity and cultural characteristics of fungal diseases of cabbage in different field sites of Santa and Dschang. Secondly, to carryout molecular identification on the main species of fungal pathogens causing diseases of cabbage and thirdly, to determine the effect of soil physiochemical properties on disease incidence and severity

A total of five cabbage leaves were collected per cabbage farm for any cabbage whose leaves showed fungal diseases symptoms in 10 farms in Santa and Dschang each making a total of 100 samples. Soil samples were also collected in regions showing no diseases incidence, low diseases incidence, moderate disease incidence and high diseases incidence to conduct a soil physicochemical analysis. The samples were transported to the Life Science Laboratory in the University of Buea for isolation and morphological identification of pathogenic fungi.

In the laboratory, the working environment was surface sterilized with 70% alcohol to reduce chances of contamination. Potatoe dextrose agar (PDA) medium with gentamycin and penicillin was prepared, the antibiotics were added to prevent the growth of other microbes such as bacteria.

For inoculation, cabbage leaves portions that showed disease symptoms were put in small nets, surface sterilised by immersing in 10 % sodium hypochlorite solution, rinsed in distilled water, rinsed with 70 % alcohol, rinsed in sterile distilled water and finally tap water, then plated. The plates were incubated at room temperature (25 °C) in the dark for 7 days. After 7 days, fungi that grew on the inoculants were sub-cultured on fresh PDA plates at room temperature. Cultural features such as colony diameter, colony colour, texture, margin, form, elevation was recorded by observing the growth forms on PDA physically. The mycelium was observed under a light microscope and crosschecked with those in literature. Out of the 100 samples, we ended up with 44 clean samples. The mycelia of these pure cultures were harvested and sent to Germany for molecular identification.

DNA extraction was done following a sorbitol-CTAB protocol and DNA purity checked in a Nanodrop. The amplified products were purified and Sanger sequencing was done by the Biocenter of the Ludwig Maximillians-University Munchen, Germany for ITS and TEF regions. Sequences obtained were edited manually in BioEdit and Chromas, after which all the sequences were subjected to Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information to identify the newly obtained sequences with those in the Genbank.

The results of the study showed that the disease incidence and severity of fungal diseases on cabbage in Santa was 1.16% and 1.76% in Dschang, showing that Dschang had a higher disease incidence as compared to Santa. So, there is a higher probability of crop failure in Dschang. These results could be as a results of the fact that most of the farms sampled were in valleys in Dschang.

The results of the disease severity for the two regions are in Tables 1 and 2. The results showed that for cabbage plants where the leaves proportion was affected up to 25% Santa had 169 while Dschang had 66. Comparing their severity with cabbage plants with about 50% leaves infected Santa had 79 while Dschang had 198. At about 75% of total leaves infected Santa had 53 while Dschang had 143 and for cabbage plants destroyed Santa had a total of 108 while Dschange had 213. The results showed that diseases severity was more severe in Dschang as compared to Santa. The results match with that of Liu et al., 2017 who reported that the most severe cabbage disease was cabbage yellow caused by Fussarium oxysporum responsible for huge cabbage losses in cabbage farms in China.

Table 1: A severity scale according to Cobine and Fuente 2015 in Santa

Table 2: A severity scale according to Cobine and Fuente 2015 in Dschang

The results of the cultural characteristics of fungal diseases of cabbage were done based on colony characteristics on culture media indicated, growth diameter, colony form, and colony colour on media both front and reverse. Out of the 100 samples that were cultured 81 proved to be attacked by fungi pathogens. The growth parameters recorded were as follows, 6 samples had circular growth margins with an ash surface colony colour and looking dark in their reverse’s colony colour. Ten samples had a raised growing form with circular surfaces and were looking green and dark in the reverse colony colour. Other species were floppy white in colour and creamy when the petri dish was turn on the reversed direction. Many of the species also show irregular growing margins and colony colony. Colours like green, white, white, green, leafy green, pink and orange. The results that were obtained in the laboratory matches with that of Hafizi et al. (2013) who recorded similar results when doing diseases isolations in common food crops. Both cultural and morphological studies are essential for the identification of plant fungi pathogens that causes disease on cabbages. The variation in the different colony characteristics was an indication that cabbage in the field is affected by a diversity of fungi pathogens.

The results based on molecular identification showed that 7 species of Fusarium oxysporum were identified belonging to the order Hypocreales and family Nectriaceae. This was the second most abundant pathogen that was isolated and only two species of Fusarium graminearum were obtained and a single species of Fusarium caucasicum. The most abundant fungi pathogen identified was Trichoderma harzianum with 13 species belonging to the order Hypocreales and family Hypocreaceae. Other species Trichoderma Sp (6 species), Trichoderma breve (1 species), Trichoderma lixii (1 species) were identified indicating that the genus was the most abundant in the samples. Four species of Curvularia senegalensis were identified belonging to the order Pleosporales and Pleosporaceae. The genus Alternaria has three species which include Alternaria sp., A. alternate and A. tenuissima belonging to the order Pleosporales and family Pleosporaceae The genus Gibberella had two species which include Gibberella fujikuroi var. moniliformis belonging to the order Hypocreales and family Nectriaceae. The genus Penicillium had only a single species which was Penicillium citrinum from the order Eurotiales and family Trichocomaceae. Another species which was identified that belong under the same order and family was Aspergillus niger. Three species were identified which belongs to the order Pleosporales and family Didymellaceae. The three species were Phoma sp., Dothideomycete sp. and Epicoccum nigrum. One species of Curvularia pseudobrachyspora was identified which belongs to the family Pleosporaceae and order Pleosporales. Lastly, Candida railenensis and Meyerozyma caribbica were identified and the two were the lone two species yeast that affect cabbage in the field

The result from soil analysis indicates that the highest pH water was 6.6 obtained in Dschang soil collected in portion with moderate disease incidence in the field while the lowest was 4.8 collected also in a portion with a moderate disease incidence in Dschang. However, generally the pH water in all the soil samples that were collected has shown that the majority of the samples has weak acidic properties.

The results when comparing the pH KCL of the soil samples collected has shown that the highest was 5.4 collected in a farm portion with a moderate disease incidence in Dschang while the lowest pH KCL was 4.1 collected in a farm portion with a moderate disease incidence in Santa. According to the results it has been seen that average pH KCL of the soil was 4.5. This indicates that there is no great variation between the soil’s pH KCL of Santa and Dschang.

The clay content of the soil indicates that the highest clay percentage was recorded in 33% at Santa in portions of moderate incidence and in Dschang in portions of no and moderate disease incidence while the least was at 30% in Santa in the portions of moderate disease incidence and in Dschnag in the portions with low and high disease incidence. The average clay content of the soils of Santa and Dschang was 31%. Also, this is an indication that the clay content of the soils in the sampled area had just the slight variation.

The results when comparing the silt content of the soil samples collected has shown that the highest percentage was 6% collected in a farm portion with low, moderate and high disease incidence in Santa and also in Dschang at no, low, moderate and high disease incidence. The average silt content results imply that both Santa and Dschang have silt in their soil which is above 5%. Hence the soils collected from the two sample areas have only minor differences with Dschang carrying the highest percentage of soil in their soil.

Comparing the quantity of sand found in the soil samples collected showed that the highest amount of sand was 66% found in the Santa in farm portions which showed no disease incidence while the lowest sand quantity was 61% as shown in Santa at low diseases incidence portions of the farm and also in Dschang at no and moderate disease incidence. However, the results indicated that the soils collected from both areas have high concentrations of sand which helps in drainage of the soil. This is an indication that the soil is good for cultivation.

I have gained a lot of experience during this period of my research thanks to the support BSPP grant. I was able to accomplish three of my specific objectives of which I could not have been able to proceed with the remaining objectives without the results of the first objective. I appreciate the BSPP for their generosity because I wouldn’t have been able to go this far within this short time with my research if the grant was absent especially as my county’s economy was greatly affected by COVID-19. I also wish to thank my supervisor Prof Tonjock Rosemary Kinge in a special way for without her, no other person would have given me the wonderful training and follow-up throughout this work and making it successful. I also give special thanks to the laboratory technician Mr Mbabe Felix and the lab assistant Mrs Moforcha Lilian for their time, patience and directives they offered to me while I was at the Life Science laboratory of the University of Buea. I am grateful to BSPP for recognising my project by awarding me with BSPP PhD bursary.

NSAH FRANCOLINE SAMA

The University of Bamenda, Cameroon

This is the report from a BSPP Lockdown Bursary.