Identification and pathogenicity of Nigrospora sphaerica and Curvularia penniseti associated with leaf spot disease of cowpea (Vigna unguiculata (L.) Walp.) in Nigeria

Abstract Pathogenic fungi are one of the main causes of plant diseases due to their ability to colonise plants and rapidly spread disease. Cowpea is one of the most highly consumed grain legumes in Nigeria, but its yearly production is usually affected by fungi attacks. Disease presenting browning of the aerial parts of the cowpea plant was observed during the dry season between 2021 and 2022 in the country. This disease caused stunted growth in the affected plant stand. The fungi were isolated from the symptomatic aerial parts. The fungi were viewed under the microscope and identified by molecular and phylogenetic tools using the Internal Transcribed Spacer (ITS) gene region. The fungi isolated from the symptomatic aerial part of cowpea were identified as Nigrospora sphaerica and Culvularia penniseti. The pathogenicity of the isolated fungi was tested on detached leaves and stems and on intact cowpea seedlings. The test gave a positive result, which confirms the pathogenicity of the isolated fungi on the cowpea plant. This is the first report of N. sphaerica and C. penniseti infecting cowpea cultivated on farmland in Nigeria. Early detection of foreign fungi causing disease in the country’s cowpea crop will aid researchers in addressing the negative effects on crop yield.


Introduction
Crop cultivation is usually affected by several factors, which could be abiotic factors such as drought, temperature, and chilling or biotic factors such as microorganisms, weeds, and insects. Among the biotic factors are microorganisms such as fungi, some of which are beneficial while others are pathogenic. Phytopathogenic fungi (fungi that cause plant diseases) cause contaminations and infections in both cultivated crops and during storage. These infections are usually observable on seeds, fruits, leaves, stems, and roots of cultivated plants, which compromise the health and yield of crops. Fungi pose the biggest economic and ecological threats among plant pathogens (Doehlemann et al. 2017). They do this by secreting mycotoxins and proteinaceous toxins, leading to cell death and pathogenicity in hosts. (Howlett 2006).
Leguminous crops are globally affected by pathogenic fungi like Cercospora canescens and Pseudocercospora cruenta, which cause "Cercospora leaf spot" in cowpea and result in about 18-42% yearly production losses in several developing countries (Pande et al. 2009). Several other diseases caused by phytopathogenic fungi in legumes are "chocolate spot disease" caused by Botrytis cinerea in faba beans (Dereje and Yaynu 2001;Berhanu et al. 2005), "legume blight disease" caused by Didymella rabiei and "wilt disease" caused by Fusarium spp. in chickpeas (Singh and Sharma 1998;Chand and Khirbat 2009), etc. Cowpea (Vigna unguiculata (L.) Walp.) is a legume crop that is widely grown and consumed in the tropical regions. The West African region is accounts for 87% of cowpea's production globally (FAO 2014), and its worldwide output is 5,249,571 tonnes (Alemu et al. 2016). Fungi diseases affect this output on a yearly basis. Most cowpea diseases are transmitted by seeds (Mamkaa and Gwa 2018), over 30 fungal pathogens have been identified in cowpea that are seed-borne (Awurum and Enyiukwu 2013). The largest producer and consumer of cowpea in the world is Nigeria, and the amount of dried grain produced in the year 2013 were 2.95 million metric tonnes, the second largest producer is Niger, followed by Burkina Faso, Myanmar and Mali (FAOSTAT 2013).
In Nigeria, Williams (1975) reported diseases of cowpea caused by several fungi, among which are anthracnose and brown blotch caused by Colletothrichum sp. in the southwest region of the country (Emechebe and Florini 1997;Adegbite and Amusa 2008). Emechebe and McDonald (1979) reported "web blight" seedling disease in the same region of the country. Diseases in cowpea result in production losses in Brazil, and lead to the 50% death of cowpea crops in Nigeria, and the United States of America (Kumar and Kalita 2017).
Some diseases were observed in cowpea cultivated on Bowen University farm land and its environs in south west Nigeria. The disease presents browning of the aerial parts of the cowpea plant with spots ( Figure 1A). Most of the earlier studies identified fungi infecting cowpea morphologically and macroscopically (Ekhuemelo et al. 2020;Deepika et al. 2021), but this approach has limitations and unnecessary species names and synonyms have been created (Bhunjun et al. 2021).
The goal of this study was to molecularly identify pathogenic fungi connected to cowpea cultivation at Bowen University in Nigeria. The specific objectives include: (1) isolation of fungi from infected leaves and stems of cowpea; (2) carry out the morphological and molecular characterization of isolated fungi from infected cowpea on Bowen University Research farm; and (3) to test the pathogenicity of the isolated fungi. Knowledge about the pathogens infecting cowpea will help design strategies to manage and prevent the diseases in the crop.

Collection of stem and leaf samples
This research was carried out at Bowen University, Iwo, Nigeria. The infected cowpea leaves and stems were collected from the Bowen University research farm Iwo and a farm land around the campus (Latitude 7°38′N 4°11′E). Samples were collected from plants showing signs of infection, that is, yellowing of leaves, leaf spots and stunted growth. Plant parts from healthy looking cowpeas were also separately collected as control. The collection of plant samples took place between December 2021 and February 2022. Average daily temperature of 24.5 ± 2 °C/33.0 ± 2 °C (night and day), relative humidity 16-33%, wind speed 2.2-4.5 mph. The samples were transported to the Plant Biology Research Laboratory in a conical flask containing distilled water to keep the cells alive within an hour of the sample collection. Collection and isolation were repeated thrice.

Isolation of fungi
Fungi were isolated from the collected infected leaves and stem samples. Firstly, the excised samples were washed with tap water to remove soil debris, and the explants (leaves and stem) were surface sterilized in 5% sodium hydrochloride for 2 min. Afterward, the explant was sterilized in 70% ethanol for a minute and rinsed three times in sterile distilled water for about 1-2 min. The edges of the explant were cut-out. Explants sized 2 mm were placed on Potato Dextrose Agar (PDA) medium containing antibiotics (gentamicin, Rashi Pharma, India). Explants from healthy plants were also surface sterilized and incubated in PDA media. The plates were incubated at 27 °C in the laboratory for 5-7 days. The fungal colony observed was separately subcultured to a new PDA (in triplicates) to have a monoculture and were observed daily for growth.

Morphological and microscopic identification of fungi
The isolated fungi were first identified morphologically and features such as growth pattern and hyphal colour were observed. A tiny portion of the individual fungi mycelia observed was gently placed on a glass surface using a sterile inoculating pin. A drop of lactophenol blue (stain) was placed on the glass-slide containing the fungal isolate. Afterwards, it was teased and a cover slip was placed over the mycelia and it was viewed with a microscope under a high power (×40) objective lens.

Molecular identification of fungi
The isolated fungi were subsequently identified using molecular methods. About 100 mg of fungus mycelia were macerated in a sterile mortal, followed by the transfer of the extract into a 1.5 ml Eppendorf tube using 1 ml of DNA Extraction Buffer (DEB) containing proteinase K (0.05 mg/ml). After adding 50 µl of 20% sodium dodecyl sulphate (SDS), the mixture was heated to 65 °C for 30 min in a water bath. It was allowed for the tubes to cool to room temperature. Potassium acetate in a concentration of 7.5 M was added and quickly mixed. This was spun for 10 min at 13,000 rpm. After gently inverting the tubes 3-5 times, the supernatant was placed into brand-new, autoclaved tubes with 2/3 volumes of cold isopropanol and incubated at −20 °C for an hour. This was then centrifuged for 10 min at 13,000 rpm, with the supernatant being discarded. After adding 500 µl of cold 70% ethanol, the mixture was centrifuged at 13,000 rpm for 5 min. The intact DNA pellet was carefully removed from the supernatant, and it was dried for 30 min at 37 °C. Re-suspended in 50 litres of sterile distilled water was the DNA pellet. The purity of the extracted DNA was further determined by detecting the amount of proteins still present in the DNA at 260/280 nm using a ThermoScientific Nanodrop spectrophotometer, model 2000.

Polymerase chain reaction (PCR) and sequencing
In this method, 10 µl of the 5x GoTaq colourless reaction, 3 µl of the 25 mM MgCl 2 , 1 µl of the 10 mM dNTPs mix, and 1 µl of 10 mol −1 each forward primer made up the PCR reaction cocktail (ITS 1) -Reverse primer, 5′ TCC GTA GGT GAA CCT GCG, and (ITS 4) − 5′ TCC TCC GCT TAT TGA TAT GC 3′; 0.24 l of 0.3 units of Taq DNA polymerase (Promega, USA) diluted in sterile distilled water and 8 l of working DNA template. Utilizing a thermal cycler for the PCR system (Applied Biosystem Inc., USA), amplification was performed using a PCR profile that included an initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 55 °C for the 30 s, and 72 °C for 1 min 30 s; and a final extension at 72 °C for 10 min.
The amplified DNA fragments of target sequences in PCR were ethanol purified in order to remove the PCR reagents. The presence or absence of the expected band size of the amplified target ITS gene sequence was checked when the purified fragment was run on a 2% agarose gel electrophoresis at a voltage of 120 V for about 1 h.

Evolutionary relationships of taxa of the two fungi isolated from cowpea
The Neighbor-Joining approach was used to infer the evolutionary history (Saitou and Nei 1987). The ideal tree is displayed. In the bootstrap test (1000 repetitions), the proportion of duplicate trees in which the connected taxa clustered together is displayed next to the branches (Felsenstein 1985). Using branch lengths in the same units as the evolutionary distances used to estimate the phylogenetic tree, the tree is rendered to scale. The evolutionary distances, which are measured in base substitutions per site, were calculated using the Kimura 2-parameter technique (Kimura 1980). Next to each internal node in the tree is the percentage of places where at least 1 unambiguous base is present in at least 1 sequence for each downstream clade. This investigation used six nucleotides. Six nucleotide sequences were subject to this investigation. Codon positions 1st + 2nd + 3rd + Noncoding were included. For each sequence pair, all unclear places were eliminated (pairwise deletion option). The final dataset contained 714 locations altogether. In MEGA X, evolutionary studies were performed (Kumar et al. 2018).

Pathogenicity test
The pathogenicity of the isolated fungi was tested using Koch's postulate assay of pathogenicity on the detached tissue (leaves and stem) and on an intact plant. Five seeds of cowpea (Landrace) were sown in 10 plastic bags and watered daily. Four weeks after emergence, seedlings were thinned to 3 stands per bag, then some of the seedlings used for further experiment.
The materials to be used for the experiment were sterilized before the pathogenicity of the fungi was determined. The isolated fungi were individually re-cultured on detached 4 weeks old leaves and stems. Whatman No. 1 filter paper pre-socked with 10 ml of distilled water was placed in a sterilized Petri dish. Fungi mycelia were picked with a 6 mm cork borer and placed on healthy detached leaves or stems in the dish, while potato dextrose agar was placed on the detached tissue (leaves and stem) as a control. The detached tissue was observed daily for the exhibition of disease symptoms. While on the intact plant, 10 ml of distilled water was poured into the plate containing the grown fungi, which was further scraped using a sterile glass rod and poured into a tube to obtain spores. The healthy cowpea plants were each sprayed with 5 ml of fungi spores, while the healthy cowpeas were sprayed with 5 ml distilled water to serve as the control.

Description of isolated fungi
On the subcultured infected cowpea leaves and stem, a fungi culture with two distinct colonies was observed (Figure 2A, B). After the fungi from the leaf and stem were separated into monoculture, the first fungus identified (labelled P2) has a black cotton appearance, with the colour of the colony initially being pinkish grey, then turning olive brown or black as the colony matures, and greyish on the reverse side ( Figure  3A). The second fungus (labelled T2) had a white cotton appearance (translucent white), which later turns brown due to mass sporulation and black on the reverse side ( Figure 3B).
The fungi were molecularly identified as Nigrospora sphaerica and Curvularia penniseti. When compared to reference sequences from the GenBank NCBI database, Nigrospora sp. showed 99.82% similarity to N. sphaerica (KU553345.1), and the sequences were deposited in GenBank (Accession number: ON568194), while Curvularia sp. showed 99.49% similarity to C. penniseti (MN889466.1), and the sequences were deposited in GenBank (Accession number ON568195). The two fungal pathogens were therefore identified as N. sphaerica strain H1 and C. penniseti stain CZPMP-21 based on morphological, microscopic and molecular features.

Phylogenetic tree analysis
Summary interpretation showed that sample P2 used in this study has 77% evolutionary similarity with N. sphaerica, accession number MF434826.1 and both showed 70% relatability with N. sphaerica, accession number, KJ767121.1. Sample T2 also showed a similar origin with C. penniseti isolates (Figure 4).

Koch's postulate assay
After day 3 of inoculating the PDA disc with test organisms on detached leaves and stems of cowpea, the fungus isolate C. penniseti exhibited disease symptoms on the leaf and stem ( Figure 5). Detached stem and leaf samples with PDA from N. sphaerica also exhibited disease symptoms after day 3 ( Figure 6).
On intact 4-week-old cowpea plants, the fungi isolate N. sphaerica and C. penniseti exhibited the browning and wilting of leaves symptoms after spraying cowpea seedlings with spore cultures (Figure 7).

Discussion
Several diseases caused by fungi in cowpea were earlier reported to be associated with periods of high relative humidity, which are characteristic of the southwestern region of Nigeria (Ajibade and Amusa 2001;Adegbite and Amusa 2008). But there is a paucity of documentation on the prevalence of fungi infecting cowpea plants during the period of low humidity and high wind in the dry season. Fungi spores are transported easily during the dry season, which encourages the easy spread of spores in the high wind and, subsequently, fungal infections in cultivated plants. In this study, N. sphaerica and C. penniseti were identified as pathogenic fungi in cowpea crops cultivated on farmland.
Due to individual differences in visual perception, fungus identification based on morphology presents difficulties for accuracy and precision (Lücking et al. 2020). Depending on the environment, fungi cultures may also display variations in their physical appearance. Hence, the use of ITS in this investigation enables impartiality in the identification of fungi.
Nigrospora sphaerica was reported to cause shot hole disease on Mulberry in China (Chen et al. 2018), and leaf spot disease on cowpea in India, which occurred between the wet periods of August and September (Deepika et al. 2021). This study provides the first evidence of N. sphaerica's involvement in the leaf spot disease of cowpea in Nigeria. Several species of Curvularia were reported to infect sorghum seeds and also cause loss of seed viability in sorghum plants in India (Girish et al. 2011). Curvularia spp. infections are common within the plant species in the grass family (i.e., Poaceae) such as maize, millet and sorghum, causing diseases such as seedling blight, root rot, etc. (Chahal et al. 1994;Iftikhar et al. 2003;Rana et al. 2022). In Nigeria, a member of the genus Curvularia pallescens was reported to cause leaf spot in maize (Mabadeje 1969). This research is the first to document C. penniseti causing leaf spot disease on cultivated cowpea in southwest Nigeria.
To positively identify a fungus as the cause of a disease, it must be isolated from subsequent specimens and tissues taken from the lesion must have fungal elements that are morphologically consistent with the isolate (Kobayashi 1996). N. sphaerica and C. penniseti isolated from the symptomatic leaves and stem of cowpea were identified as the fungi causing infection and when introduced into healthy cowpea plants as stated by Koch' postulates of the pathogenicity of microorganisms, the infectivity of the two fungi was observed.
The occurrence of pathogenic fungi infesting legume crops is associated with factors such as climate change (Pandey and Basandrai 2021). Elevated temperature, which affects soil moisture, contributes to the spread and emergence of new plant diseases (Sharma and Ghosh 2017), resulting in the presence of this new species of fungi in the studied area. An earlier report on the pathogenicity of C. penniseti stated its effects on seedling mortality and delayed germination in pearl millet seeds (Singh et al. 2008).
The two pathogenic fungi in this study present a threat to the cultivation of cowpea in this region. Therefore, there is a need to find appropriate control measures. Biodegradable fungicides, such as plant extracts, have proven to be effective in combating fungal infection in cowpea seedlings (Kareem et al. 2018;Obisesan et al. 2022). This could be employed in combating the effects of this infection in cowpea.

Conclusion
Pathogenic fungi are known to cause major diseases in plants and are affecting crop yield. Fungi have been associated with yield reduction in cowpea and several measures have been taken that are being used by farmers to solve this problem. With changes in climatic conditions, new fungi are usually introduced to the farm. In this study, two fungi N. sphaerica and C. penniseti associated with leaf spot appearance in cowpea were identified and documented for the first time in Nigeria. The pathogenicity of this fungi was also established.