Choanephora cucurbitarum (Mucorales) is an important plant pathogen that causes soft rot of floral parts and fruits of many plants, including summer squash, pumpkin, pepper and okra (Agrios 1997). Cauliflower (Brassica oleracea) is an important crop in India and any diseases found on this host are of potential agricultural and economic relevance.

In September 2012, 2013 and 2014, leaves of an early variety of cauliflower (cv Early Kuary) bearing typical soft rot symptoms, accompanied by superficial fungal growth were collected at Palarikhurd Village, Sonipat (28°59′26.53″ N; 77°00′57.30″ E; 249 m a.s.l.), Haryana, India. Disease symptoms were observed and photographed in the field. Samples were taken for later examination in the laboratory. Fragments of infected leaves bearing pin head-like fungal growth were surface sterilized by dipping in 1 % sodium hypochlorite for 1 min and rinsing once with sterilized distilled water and aseptically transferred to plates containing PDA (potato dextrose-agar) and left at room temperature for 48 h. Homogeneous cultures were obtained and one was selected for further study and deposited in the Indian Type Culture Collection (ITCC 7521).

Slides were prepared by mounting a colony fragment in lactophenol. The fungal structures were observed under a microscope (Olympus, Magnus MLXi). The morphological and cultural characters of the fungus were recorded and compared with published descriptions of soft rot-fungi (Kwon et al. 2001; Kagiwada et al. 2010). Profuse and rapidly growing white mycelium was formed on PDA after 36 h. Sporangiophores bearing apical sporangiola were formed. Sporangia were sub-globose (35 – 85 μm diam) and monosporus sporangiola were elliptic, fusiform or ovoid (8–13× 11–22 μm) (Fig. 1c). Sporangiospores were elliptic, fusiform or ovoid (7–10.5 × 10-27 μm), brown, with or without three or more thin appendages at both ends (Fig. 1d). This morphology is typical of C. cucurbitarum as described by Kwon et al. (2001) and Kagiwada et al. (2010).

Fig. 1
figure 1

Symptoms and signs of Choanephora rot on cauliflower and morphology of the causal agent Choanephora cucurbitarum a Symptom of leaf rot of cauliflower in field; b Numerous monosporus sporangiola (at 10 x 100X); c Fusiform sporangiospores with appendages (at 10x100X); d Cauliflower seedlings showing typical symptoms of leaf and bud rotting

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Identity of the fungus involved on cauliflower leaf rot was further investigated by comparison of ITS and TEF sequences of ITCC 7521 with GenBank data (White et al. 1990).

Genomic DNA was extracted from the pure culture of C. cucurbitarum through the cetyl trimethyl ammonium bromide (CTAB) method with slight modifications (Karthikeyan et al. 2010). DNA extracted by adding chloroform: isoamyl alcohol in the ratio of 24:1centrifuged at 12,000 rpm/10 min repeated this step twice, suspended with 150 μl of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). RNA was digested by adding 2 μl of RNAse A and incubated at 37 °C for 1 h and stored −20 °C for further use.

The internal transcribed spacer (ITS) region was amplified with universal primers ITS1 and ITS4 (White et al. 1990). The reaction mixtures were prepared in a total volume of 100 μl with a final concentration of 50 ng DNA, 10 μl of 10 × assay buffer, 1.5 μMMgCl2, 10 μM dNTP mix, 10 pM of each primer, and 2.5 U of Taq DNA polymerase (Bangalore Genei, India) and the volume was made up to 100 μL with sterile double distilled water. The PCR was performed with a PCR Thermal Cycler MP (Bioer, Japan) according to the following program sequence: initial denaturation 94 °C for 3 min, followed by 35 cycles of 1 min at 94 °C, 30 s at 60 °C and 1 min at 72 °C with a final extension step for 3 min at 72 °C. After thermal cycling, the amplified product was run on a 1.2 % agarose gel, stained with ethidium bromide, The gel was visualized under UV radiation in a gel documentation system (Biorad, USA) and digitally photographed (Canon,Tokyo, Japan).

Translation elongation factor (TEF) was amplified with specific primers EF1-645F and EF1-1190R (Fiers et al. 2011). The reaction mixture prepared to the total volume of 50 μl with final concentration of 75 ng of DNA, 5 μl of 10X Taq buffer, 1.5 μM MgCl2, 8 μM dNTP mix, 10 pM of each primer, and 1.5 U of Taq DNA polymerase (Bangalore Genei, India) and the volume was made up to 50 μL with sterile double distilled water. The PCR was performed with aPCR Thermal Cycler MP (Bioer, Japan) according to the following program sequence: initial denaturation 94 °C for 2 min, followed by 30 cycles of 1 min at 94 °C, 30 s at 56 °C and 1 min at 72 °C with a final extension step for 10 min at 72 °C. The amplified product was electrophoresed and digitally photographed as cited above.

The amplified products of ITS and TEF were partially sequenced at Amnion DNA sequencer (Bengaluru, India). The sequences were deposited in NCBI GenBank under the following accession numbers (for ITS - KJ817815, for TEF- KM214205). A BLAST search in GenBank revealed 99 % NT identity with C. cucurbitarum as per the comparison with the accession numbers KF724997 for ITS and AF157239.1 for TEF. Identity of the isolate ITCC 7521 was therefore confirmed as C. curbitarum.

Healthy seedling of cauliflower (cv Early Kuary) were planted individually in each of ten plastic pots containing containing sterilized sandy loam soil. When the plants were thirty days-old they were inoculated with 5 mm diam, sporulating culture discs obtained from 5 days old ITCC 7521 colonies formed on PDA. Discs were placed on leaf margins (two leaves/plant) and tender buds. The pots were then covered with an internally moistened plastic bag and kept inside an incubator (Ferrotek) adjusted to 25 °C. The plastic bags were removed after three days. As a control, PDA disks with no fungus were deposited on leaves of two separate plants and these were treated as described above. Typical soft rot symptoms appeared after 5 to7 days of inoculation, firstly at the margins of younger leaves as water soaked areas, followed by inward rolling and rapid rotting of the infected leaves that became black (Fig. 1f). Later (7 days after inoculation) the infected areas were covered with pale greyish brown fungal colonies (Fig. 1g). This is the first record of C. cucurbitarum causing leaf rot of cauliflower in India and the first time the pathogenicity of this fungus to cauliflower was demonstrated.