Introduction

Cowpea, Vigna unguiculata (L.) Walp. (Fabaceae) is an important staple food for millions of people in the arid and semi-arid tropics [3, 25]. It has been estimated that about 3.3 million metric tonnes of cowpea produced worldwide during 2000. The global production of cowpea is up to 5.59 million metric tonnes. The Western Africa contributes cowpea production up to 81% followed by Eastern Africa (8.68%) and Central Africa (4.37%) [1, 14, 33]. Cowpea is also a chief grain legume in the sub-tropics as food and forage in sub-Saharan Africa [9, 32]. The grains of cowpea besides providing proteins and carbohydrates, its tender leaves are also edible [23, 28, 33]. Cowpea serves as potential nutritious fodder for the livestock. It is a resourceful crop owing to restoration of nitrogen content in soils leading to enhancement of soil fertility to grow cereals as an alternate crop [5, 10, 21, 24].

In India, pulses have been regarded as source of protein and they play an important role in healthy diets, sustainable food production and in larger context the food security. India has produced 23.40 million tonnes of pulses during 2018–2019 crop year yet we import 26–28 million tonnes to meet the national requirements [2]. Karnataka State is one among the main producers of pulses. The area under cowpea production in Karnataka state is increasing, there are several constraints allied with cowpea production as a result we are experiencing a large quantity of production loss. A large number of plant diseases and pests are considered as major constraints to cowpea production and they affect the crop at various stages of growth resulting in significant yield loss [29, 31]. The important diseases threatening the cowpea production includes anthracnose, blights, charcoal rot, collar rot, leaf spots, rusts, powdery mildew, root rot, southern blight, and others.

Recently, various researchers have reported the incidence of root rot/dry root rot diseases in cowpea caused by F. equiseti [17], F. oxysporum, F. proliferatum [26, 27] in the United States; target leaf spot disease by Corynespora cassiicola in China [16], leaf spot diseases by Pestalotiopsis sp., Dactuliophora sp., and collar rot by Aplosporella hesperidica [6, 18, 19] in Southern India. Dactuliophora sp. was considered causing minor leaf spot disease of cowpea in Tropical Africa [30]. However, the disease incidence and severity reported in this paper denotes that Dactuliophora sp. causes a major disease in Southern India. The field survey was carried out to determine the occurrence of the zonate leaf spot (ZLS) disease. Therefore, the present study aims at detailed study of its morphological and cultural features to establish and to document the pathogen identity, extent of distribution and severity of disease in southern districts of Karnataka, India.

Materials and Methods

Survey of Disease

During 2016–2018, the major cowpea producing regions of Mysore district of Karnataka State were visited and the occurrence of ZLS disease was observed in many fields. The disease incidence (DI) and per cent disease index (PDI) was estimated following the procedure proposed by Mahadevakumar et al. [20]. The survey was carried out during months of August–November in a farmers’ field in main cowpea growing locations. Disease Incidence was assessed by expressing the number of ZLS diseased cowpea plants. To record the disease in an infected cowpea field, 5 m2 area each at four corners and the centre (total, 5 plots) were selected. On each plot, a number of healthy plants and infected plants were recorded. Further, to measure the PDI, all the infected leaves were categorized into different grades of infection using the following grading scale: 0 = no infection; 1 = 1–5%; 2 = 6–25%; 3 = 26–50%; 4 = 51–75%; and 5 = 76–100% leaf lamina covered by infection [20]. The incidence and severity were the mean value of leaf spot infection of 3-year assessment (data expressed in percentage). The diseased leaf samples were collected for further investigation. The fungal pathogen was isolated from the infected leaves.

Isolation and Identification

Infected cowpea leaves were cut into 1 cm2 pieces, surface-sterilized (sodium hypochlorite, 2%) followed by rinsing thrice with sterile distilled water. The leaf pieces were blotter dried and inoculated on the surface of potato dextrose agar (PDA) medium. After incubation up to 5 days, fungal colonies developed from the infected leaf pieces were subcultured on fresh PDA medium to obtain pure cultures. The morphological features of the pathogen on the host plant and its cultural characteristics on the medium were considered for identification [4, 8]. A censorious examination of the symptoms and sclerotial characteristics by micrometry were considered for comparison with the Dactuliophora tarrii to characterize the new pathogen.

Pathogenicity Test

Pathogenicity tests were performed on healthy cowpea plants under green house conditions to ascertain the association of the isolated fungus causing ZLS disease. The experiments were conducted on 45 days-old healthy cowpea plants with 15 days-old suspension of Dactuliophora sp. [18]. The inoculated plants were kept under high humidity (80%) for 5 days and at the ambient conditions. Cowpea plants inoculated with distilled water served as control. The experiments were carried out in triplicates with two repetitions to confirm the pathogenicity and successful manifestation of symptoms on the host plant similar to naturally infected ones.

Molecular Identification

The genomic DNA was isolated using 10-day-old fungal cultures using the CTAB method [36]. The PCR was performed using Applied Biosystems Veriti Thermocycler (Foster City, CA, USA). The ITS-rDNA region was amplified employing ITS1 and ITS4 universal primers [35]. The PCR reaction was carried out in 25 µL reaction mixture containing 2 µL of DNA sample, 12.5 µL of ready-mix (Genei, Bangalore, India), 20 pmol of each forward and reverse primers (1.0 µL) (Sigma, Bangalore, India) and the final volume was made up to 25 µL with 8.5 µL of nuclease-free water. The PCR programme include initial denaturation (95 °C, 5 min) followed by 38 cycles of denaturation (94 °C, 1 min), primer annealing (55 °C, 30 s), extension (72 °C, 2 min) and the final extension (72 °C, 12 min). The amplified PCR products were sequenced using an ABI3730 x I DNA analyzer (Applied Biosystems, Foster City, CA, USA). The representative reference sequences were retrieved from the NCBI-GenBank Database. The DNA sequences were nBLAST searched against the NCBI nucleotide database for closely matching relatives. A phylogenetic tree was constructed for further confirmation of the species in a combination of DNA sequences of our isolates and the reference sequence from the GenBank database. The reference sequences were retrieved from the GenBank database and phylogenetic tree was constructed using the Neighbour-Joining (NJ) method as implemented in MEGA 7.0 with Kimura-two-parameter model with 1000 bootstrap replications [13].

Sensitivity to Fungicides

In the present study, three systemic fungicides viz., Thiophanate-Methyl, Benomyl and Carbendazim; one contact—Mancozeb and one combi product—Mefenoxam, were used (Table 1). All the five fungicides were tested at three different concentrations (100, 150, 200 mg/L) using the poisoned food technique [22]. The requisite quantity of each fungicide was dissolved in PDA medium after autoclaving and well grown 7 day-old culture of Dactuliophora sp. (0.7 cm diam disc) was placed at the centre of PDA plates. The PDA medium with sterile distilled water served as control and plates were observed for the growth of fungi. On the day 5, the radial growth of the fungal colony was recorded and the per cent growth inhibition was calculated: I = C − T/C × 100 (where, I, per cent inhibition; C, growth in control; T, growth in treatment) [34].

Table 1 Fungicides used for in vitro evaluation against Dactuliophora mysorensis
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Statistical Analysis

The data on in vitro efficacy of fungicides were analysed statistically with One-Way ANOVA @ P < 0.05 level by using SPSS Inc. 16.0 and treatments means were separated by Tukey’s Honestly Significant difference (HSD) tests.

Results

Diagnosis and Incidence of Disease

During the survey, the disease incidence (DI) of a new leaf spot disease was found with characteristic symptoms distinguishable from other diseases. The ZLS disease recorded on cowpea displayed characteristic rosette-like patches with whitish to tan spots on the upper leaf surface and dark-reddish brown bearing sclerotia attached through sclerotiophores on the lower surface. Leaf spots are zonate on upper as well as on the lower side of the leaf usually up to 2–3 cm in diameter, rosette-like with alternating whitish and tan bands on the upper leaf surface and light-brown on the lower leaf surface with sclerotiophores bearing dark-brown sclerotia. These sclerotia are the means of perennating structures which disseminates to considerable distance (Fig. 1).

Fig. 1
figure 1

Zonate leaf spot (ZLS) disease of cowpea caused by Dactuliophora mysorensis: a, b typical ZLS symptoms observed on cowpea; c, d close view of ZLS spot—upper surface on cowpea; e lower surface of ZLS disease of cowpea showing sclerotia developed on the necrotic lesions; f, g stereo view of ZLS disease showing sclerotia developed along with sclerotiophores on the necrotic lesion (Scale = 5 mm); h, i pure cultures of D. mysorensis and microsclerotia on PDA medium

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The prevalence and severity of Dactuliophora species infected cowpea were evaluated in 17 farmers field in and around Mysore district. The ZLS was recorded only in four locations and the incidence ranged from 8 to 22% and the PDI ranged from 1.8 to 4.6, 1.1–6.8 and 0.8–6.3 during the years 2016–2018, respectively (Table 2). The prevalence of ZLS disease was severe in Doddamaragowdanahally village (Mysore, Karnataka State, India) up to 81.39%. The details of disease incidence and PDI of ZLS disease during the survey are presented in Table 2.

Table 2 Prevalence, disease incidence (DI) and severity of zonate leaf spot disease in different locations of Karnataka, India
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Isolation and Identification

The fungal pathogen associated with ZLS disease was isolated on PDA medium. Mycelia submerged, hyaline, diffused throughout the leaf tissue and accumulated in plectenchymic masses beneath the sclerotiophores. Sclerotiophores were erumpent and shaped a ring of dark coloured hyphae, which are consistent with a sub-epidermal plectenchymic mycelial mass. The sclerotia were scattered on abaxial surface of the infected leaves, solidly hypophyllous, circular to subspherical, measured 95–185 µm in diameter bearing 4–9 scattered setae. The sclerotial setae were cylindrical, often sinuate, measured 190 µm long, 3–4 µm width and marginally constricted distally to shape a blunt tip and setae with 0–3 septa (Figs. 1, 2). On PDA medium, the growth rate was 1.15–1.95 mm/day at 27 °C. Development of profuse sclerotia were seen, those were glomerate clusters scattered over the colonies. The aerial mycelia were tan and pale-brown underneath. Sclerotia developed on well differentiated sclerotiophores like those in live host plants. A comparative account of D. tarrii and D. mysorensis is provided in Table 3.

Fig. 2
figure 2

Sclerotia and mycelium of Dactuliophora mysorensis under compound microscope: a–d sclerotia developed on host tissues observed under compound microscope showing the characteristic nature of long setae on the surface; e–h hyphae and mycelium of D. mysorensis

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Table 3 Comparison of diagnostic features of Dactuliophora tarrii and D. mysorensis (CDA, Czapek Dox agar; potato carrot agar)
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These symptoms were persistent throughout the crop season and affected the overall yield of cowpea with sclerotia forming on the lower surface. On the lower leaf surface, sclerotiophores bearing sclerotia were produced from the immersed mycelium (Fig. 3). Sclerotia were generally globose to irregular in shape and dark to grey brown in colour measuring 139.5 × 89 µm. Upon germination, germ tubes were produced over the entire surface of the sclerotium measuring 69.9 × 6 µm.

Fig. 3
figure 3

Phylogenetic analysis by Neighbour-Joining method of Dactuliophora mysorensis based on ITS-rDNA with reference sequence from GenBank database. The tree is rooted to Botryosphaeria dothidea (Tamura-Nei Substitution model and nearest neighbour-interchange search options with 1000 bootstrap replicates were used)

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The pathogenicity test revealed that, the fungus was pathogenic on healthy cowpea plants and produced a characteristic ZLS disease after 18 days of post-inoculation. The initial symptoms appeared after 15 days of post-inoculation and became prominent later with characteristic zonate spots on leaves.

Taxonomy

Dactuliophora mysorensis Deepika, Mahadevakumar, Amruthesh and Lakshmidevi

Mycobank # MB836147; Figs. 1 and 2

Holotype: UOM 2020-DM1, Herbarium, Department of Studies in Botany, University of Mysore, Karnataka, India.

Host: Vigna unguiculata (L.) Walp.

Distribution: The pathogen Dactuliophora mysorensis was recorded in different regions of the Mysore District. Geographical regions of Mysore District (Karnataka State, India) comprise dry deciduous regions with average rainfall in winter and the highest temperature recorded during summer (31 °C).

Etymology: The species named as mysorensis to assign and to commemorate the geographic location from where this species is collected and documented as new record from India on cowpea.

Type specimen: Holotype is available in the Herbarium, Department of Studies in Botany, University of Mysore, Mysore, Karnataka State, India.

Molecular Detection

The genomic DNA isolated from the pure cultures and ITS-rDNA was amplified using ITS1-ITS4 primers. The sequence analysis by nBLAST search revealed that the ITS-rDNA sequences shared 100% sequence similarity with Macrophomina phaseolina (FJ415067.1, MK573366.1, GU046874.1) and 99.83% with Rhizoctonia bataticola (KX270356.1, KX270355.1, DQ339102.1, DQ222239.1). The ITS-rDNA sequences from the present study are deposited in the GenBank database with accession # KC568286.1 and KC568285.1. Further, phylogenetic analysis revealed that the sequences shared a common clade between M. phaseolina and R. bataticola as depicted in phylogenetic tree (Fig. 3). There are other Macrophomina species placed on the same clade based on ITS-rDNA sequence, which indicates that the species though they share 100% sequence similarity, they distinctly differ in morphology and spore features.

Sensitivity to Fungicides

Efficacy of fungicides were tested in vitro for the fungal growth inhibition of the pathogen (Table 4). No growth was recorded in all three concentrations (100, 150 and 200 mg/L) of Benomyl and Carbendazim (100% of inhibition of mycelial growth), followed by Thiophanate-methyl at 200 mg/L with inhibition up to 70.93%. Similarly, 52.99% and 58.97% growth inhibition offered at 100 mg/L and 150 mg/L of Thiophanate-methyl, respectively. Metalaxyl-M showed 31.33%, 33.9% and 39.6% in 100, 150 and 200 mg/L concentration, respectively. Compared to other fungicides, the Mancozeb showed the least mycelial growth inhibition: 15.88%, 22.26% and 26.84% at three increasing concentrations (100, 150 and 200 mg/L) tested, respectively (Fig. 4).

Table 4 Effect of different fungicides on the mycelial growth of Dactuliophora mysorensis
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Fig. 4
figure 4

In vitro efficacy of fungicides evaluated against Dactuliophora mysorensis at 100, 150 and 200 mg/L concentration: a–d Thiophanate-methyl (Roko); e–h Metalaxyl-M (Ridomil Gold); i–l Mancozeb (Indofil M-45) showed varied degree of inhibition. m, n Benomyl (Benofit), and o, p Carbendazim (Bavistin) showing 100% growth inhibition at lowest concentration tested (100 mg/L)

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Discussion

Emerging diseases caused by the fungi and fungi-like organisms being increasingly reported in many crops [11]. The emerging and new fungal diseases on cowpea caused by fungal pathogens are becoming major constraints in production of cowpea. A survey conducted in the Mysore District of Karnataka (India) revealed that cowpea was infected with leaf spot disease. The infection becomes severe after rain, leading to substantial death of plants. The disease was first found during September 2010 and initially, spots were appeared as large lesions with concentric rings with whitish on the upper leaf surface and pinkish-grey on lower leaf surfaces. These symptoms were persistent throughout the season with production of sclerotia on the lower surface. Sclerotia were generally globose to irregular, dark to grey brown. Sclerotiophores develop from the mycelia immersed in the leaf tissue and produce sclerotia. Sclerotia germinate by the germ tubes over the entire surface [18]. Leakey [15] performed a comparative study to distinguish the isolated species Dactuliophora tarrii from the Tropical Africa. Although the occurrence of Dactuliophora sp. associated with ZLS disease of cowpea from Mysore region was reported earlier, there was no clarity on its species identity. The pathogen was isolated from the Mysore District in India are distinct from that of D. tarrii recorded by CLA Leaky during 1964. Based on differences in morphology with D. tarrii, the fungus isolated in the present study was recorded as new species and named as Dactuliophora mysorensis.

The ITS-rDNA sequence analysis revealed that the fungus shared 100% similarity with R. bataticola and M. phaseolina as per phylogenetic placement as well as nBLAST analysis. It is very clear that the fungus may be genetically related to R. bataticola and M. phaseolina. The taxonomic placement of mycelia sterilia is solely based on morphological features and may or may not be substantiated by sequence analysis. However, as previously predicted the relationship between R. bataticola and M. phaseolina as they are similar genetically and differ morphologically (former represent the sclerotial form and the latter representing the pycnidial form). The R. bataticola and M. phaseolina are recognized as two asexual sub-phases of which, the former has been known to form microsclerotia and the latter, forms pycnidia on host tissues and microsclerotia on culture [7, 12]. This scenario illustrates that the genetic relatedness between M. phaseolina and R. bataticola (although both are same, where M. phaseolina is pycnidial form and R. bataticola is sclerotial form) exhibits similar relationship, where Dactuliophora may represent sclerotial form of M. phaseolina or related species.

The fungicides used in the present study are generally advised to manage various fungal diseases like anthracnose, powdery mildews, leaf spot and fruit rots of vegetables and pulses (https://www.agritech.tnau.ac.in). The Benomyl, Mancozeb and Carbendazim are usually applied to treat various fungal diseases associated with vegetables, flower crops against sheath blight, loose smut, leaf spots and powdery mildew of pulses, oil seeds and vegetables (https://agritech.tnau.ac.in/crop_protection/pdf/6_Major_use_fungicides.pdf; [6]). Among the five combinations of fungicides evaluated, Benomyl and Carbendazim performed well by 100% growth inhibition followed by Thiophanate-methyl (71% inhibition). Further work needs to be carried out to evaluate the efficacy of systemic fungicides in field trials to confirm the strategies of disease management.

The new ZLS disease caused by D. mysorensis described from Southern India is distinct from the previously described D. tarrii from Africa. In order to have a better comprehension of the host range for D. tarrii, the host details available from IMI herbarium were retrieved and compiled in Table 5. The D. tarrii has been reported on various pulse crops from Africa region including Sudan, Uganda, Tanzania, Nigeria, Zambia, Zimbabwe and Malawi. Important pulses recorded to be associated with D. tarrii are Vigna unguiculata, V. coerulea, V. mungo, V. sesquipedalis, V. vexillata, Phaseolus aureus, P. lunatus and Crotalaria juncea. The D. mysorensis identified and isolated from the naturally infected leaf samples of cowpea was pathogenic and successful manifestation of symptoms were established in the plants inoculated with pure cultures in green house conditions. Since the D. mysorensis showed distinct morphology and cultural features, the erection of new species is justified. Management of the newly emerged disease is necessary to reduce the loss of production of protein-rich pulse. In the present study, the performance of systemic fungicides was better compared to contact fungicides against Dactuliophora sp. The Benomyl and Carbendazim were proved to be the most effective fungicides for complete inhibition of Dactuliophora sp. All the three concentrations of Mancozeb were less effective, while the performance of Metalaxyl-M was poor. The Benomyl and Carbendazim were effective against Dactuliophora sp. than the Mancozeb, Thiophanate-methyl and Metalaxyl-M. The Benomyl and Carbendazim may be recommended in field trials for the management of ZLS disease of cowpea caused by Dactuliophora species.

Table 5 World collection of Dactuliophora and their locality with voucher details
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