Isolation and Molecular Identification of some Fungi Associated with Jatropha curcas (L.)

Jatropha curcas is a plant of great economic importance that experiences high incidence of fungal attack. Misidentification of the fungal species is bound to occur with the use of traditional cultural methods where organisms are identified morphologically and/or microscopically. This study was carried out to isolate and identify the fungi associated with Jatropha curcas (L.) using both traditional/ cultural techniques and molecular methods. The fungi were isolated from diseased leaves and stems of J. curcas using both Standard Blotter and Potato Dextrose Agar (PDA) methods. DNA was extracted from the fungal isolates using Zymo Fungal/Bacteria DNA MiniPrep Kit. Amplification of the Internal Transcribed Spacer (ITS) regions of the fungal isolates was carried out using fungi universal primer pairs for ITS4 and ITS5. The amplicons were sequenced and the isolates were identified as Penicillium brevicompactum, Aspergillus sp., Botryosphaeria rhodina, Aspergillus nomius, Aspergillus tamarii, Rhizopus oryzae, Penicillium citrinum and Fusarium solani. Phylogenetic analysis was carried out to know the relationship between the isolates and other closely-related species in GenBank. Jatropha curcas is colonized by many fungal species some of which may be pathogenic to the plant, and molecular techniques pose the best alternative for accurate identification of these organisms.


Introduction
Jatropha curcas (Linn.), commonly called "Jatropha" or "physic nut", is a droughtresistant multipurpose shrub that originated from Mexico and Central America (Salvador-Figueroa et al., 2015). Today, the plant is cultivated world-wide as a hedge crop and for traditional purposes in both tropical and subtropical regions (Agbogidiet al., 2013).
Jatropha is a plant with many economic values. The unique oil extracted from the seeds can be used as a feed stock for biodiesel (Maghulyet al., 2013). The seed cake serves as inorganic manure and when derived from a non-toxic variety of Jatropha curcas, it may be used as animal feed. The leaves, bark, shoot latex, roots and seed oil are used for medicine and veterinary purposes. In the rural areas in Nigeria such as Aluu, Omuigwe, in Ikwere LGA; and Rumuji, Oduoha in Emohua LGA both in Rivers State, Jatropha leaf is believed to aid in fermentation of cassava and is used for this purpose. After peeling and soaking the cassava, Jatropha leaves are placed on top of the cassava and this reduces the number of days required for the cassava to ferment. The fruit exocarp (coat), seed shell and the press cake of the seed are rich in nitrogen, phosphorous and potassium and can be used as an inorganic fertilizer to improve plants growth (Islam et al., 2011).

Study Area and Sample Collection
The study was carried out at the Regional

PCR Amplification and Sequencing
Fungal universal primer pair ITS4, forward: (5´-TCCTCCGCTTATTGATATGS-3´) and ITS5, reverse: (5´-GGAAGTAAAAGTCGTAACAAGG-3´), were used to amplify the ITS1-2 region of the isolates. The PCR cocktail consisted of 3µL of genomic DNA (10ng/µL), 0.1µL of Taq polymerase, 2.5µL of 10X PCR buffer, 1.0 µL of DMSO, 1.0µL of 2.5mM DNTPs, 1.0µL of 25mM MgCl2(Promega), 1.0µL of each primer (concentration of 5μM) and 13.4µL of Nuclease-free water, making a total volume of 25µL. Amplifications were performed in a thermal cycler (GeneAmp® 9700 PCR System, Applied Biosystems, California, USA) using an initial denaturation step of 94°C for 5 minutes, followed by 36 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds and elongation at 72°C for 45 seconds with a final extension at 72°C for 7 minutes. The amplicons were stored at 10°C. Amplified products were sequenced on ABI 3500 Genetic Analyzer (Thermo Fisher Scientific, Massachusetts, U.S.A).

Data Analysis and Phylogeny
The generated raw sequences of the isolates were edited to remove PCR artifacts and sequencing noise using Molecular Evolutionary Genetics Analysis (MEGA) software, version 10.0.1 (MEGA X) (Kumar et al., 2018). A BLAST n search was conducted using the edited sequences against the GenBank database at the National Centre for Biotechnology Information (NCBI) database for identification of the isolates. A multiple sequence alignment of the sequences obtained from the search query of the isolates was carried out using Clustal W program and a neighbour-joining phylogenetic tree was constructed using maximum composite likelihood method. Other data obtained from the study were analyzed using One-way Analysis of Variance (ANOVA) (P<0.05) with the aid of Statistical Package for Social Science (SPSS) software version 23.0

Fungal Species Isolated from Jatropha curcas
Based on our morphological characterization on PDA plates ( Figure 1 and Table 1), eight fungal species were isolated from the leaves and stems of Jatropha curcas. The frequency of occurrence and the morphological characteristics of the isolates are presented in Table 1. For the organisms isolated from the leaves, sample 2 had the highest mean occurrence (42.00), followed by sample 8 (37.00), sample 7 (28.66), sample 3 (25.00), sample 5 (21.00), sample 6 (18.00), sample 4 (12.00) and sample 1 (10.33). For organisms isolated from the stem, sample 8 had the highest mean occurrence (50.00) followed by sample 2 (41.00), sample 7 (31.00), sample 3 (26.66), sample 5 (21.33) and lastly sample 6 (18.00). Sample 1 and 4 were not found in the stems. More fungal isolates were obtained from the leaves than from the stems of the J. curcas trees sampled. Pure cultures of the fungal isolates are presented in Figure 1.       (Figure 3). The tree enriches our understanding of how the fungal species evolved; which organisms evolved before the other. The greater the length of the vertical lines on the tree, the more the difference between the organisms.   (Tam et al., 2014). Aspergillus tamarii is an endophytic fungus that could be a potential source of antibacterial agents. Ogboleet al., (2017) reported the antibacterial activity of Aspergillus tamarii against Salmonella typhi, Staphylococcus aureus, Bacillus subtilis and Escherichia coli. A. tamarii has also been reported to produce the enzyme, tannase which is used in the food industry (Costa et al., 2008). Aspergillus tamarii has been found to be pathogenic to man. The production of aflatoxin B1 and G1 by A. nomius strain ASR3 has also been reported by Da Silva-Junior et al. (2017). Penicillium citrinum is known to produce a mycotoxin called citrinin. P. citrinum has been recorded as one of the seed-borne pathogenic fungi in common bean (Phaseolus vulgaris) (Marcenaro and Valkonen, 2016). P. citrinum has also been connected with the promotion of plant growth and mitigation of the adverse effects of stem rot (Muhammad et al., 2015). Penicillium brevicompactum and. P. solitum were reported as pathogenic organisms on pear (Louw and Korsten, 2014 Kurniawati and Sardjono, 2014). R. oryzae has also been reported to produce lactic acid which is widely used in food and food-related products (Kurniawati and Sardjono, 2014 and as the causal agent of crown and root rot of strawberry crops in Southwestern Spain (Pastrana et al., 2014).

Conclusion
In this study, eight fungal organisms associated with Jatropha curcas were isolated using traditional cultural techniques and identified using molecular methods. The use of traditional cultural techniques in the identification of microorganisms is not reliable as misinterpretation and misidentification of organisms abounds. The cultural techniques used in this study only aided in the processes that led to the correct identification of the isolates. The morphological characteristics were only able to suggest the suspected organisms but cannot be used to successfully characterize the isolates to the species level. Identification of the fungal isolates by the use of molecular techniques such as Polymerase Chain Reaction amplification and sequencing of the ITS regions of the fungal genome yielded a good result. Some of the organisms isolated in this study exist as endophytes, having a symbiotic relationship with plants.
Under stress or unfavourable conditions, endophytic organisms become pathogenic causing deterioration on plants. These fungi cause deterioration of the seeds, leaves and bark (or stem) of Jatropha curcas plants leading to a decrease in the quantity and quality of the plant products obtained. This study has provided information on some of the fungal organisms associated with Jatropha curcas which will help in proffering prevention and control measures to reduce the incidence of some of these pathogenic organisms on J. curcas.