Characterization of Sclerotium rolfsii causing foot rot: a severe threat of betel vine cultivation in Bangladesh

Abstract

The development of the foot rot disease caused by the fungus Sclerotium rolfsii is one of the primary variables endangering betel vine production in Bangladesh. Consequently, with the ultimate objective of finding efficient preventive and control strategies for this infamous phytopathogen, the current study was undertaken for comprehensive population structure analysis, exploration of physiological features and incidence patterns of pathogenic S. rolfsii isolates. We discovered 22 S. rolfsii isolates from nine northern districts of Bangladesh. Mohanpur (51.90%), Bagmara (54.09%), and Durgapur (49.45%) upazilas in the Rajshahi district had the more severe occurrences of foot rot disease, while Chapainawabganj (18.89%) had the least number of cases. The isolates differed substantially in terms of morphology and growth rate. By employing the UPGMA algorithm to analyze the combined morphological data from 22 S. rolfsii isolates, these isolates were divided into six different groups with a 62% similarity level. Somatic incompatibility was also found in some isolates. The RAPD-4 primer confirmed 100% polymorphism among these isolates, and these genetic variations were further validated by molecular analysis. The results of the morphological and molecular analysis revealed that there was significant variation among the S. rolfsii isolates. Finally, a comprehensive characterization of S. rolfsii would allow for a suitable management strategy for betel vine’s deadly foot rot disease.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03890-8.

Introduction

Betel vine (Piper betle L.) is a commercially valuable cash crop in Indian sub-continent including Bangladesh. It is extensively cultivated in the Indian subcontinent including India, Bangladesh, Sri Lanka, and Thailand for local consumption and export (Mahfuza et al. 2020). In Bangladesh, betel vine (local name: pann) is the main source of income for resource-poor farmers, especially in the northern districts (Ullah et al. 2020). Among the betel vine cultivating regions in Bangladesh, the Rajshahi district is one of the best-known regions for producing a special type of betel vine locally called ‘Bangla pann’ (Shimul 2021). Farmers of this region that cultivated betel vine significantly contribute to the national demand and for export abroad. Because of disease and pest problems, unavailability of the credit facilities and uncontrolled marketing the cultivation of betel vine is gradually reducing day by day. Betel vine is highly vulnerable to diseases especially against the fungal diseases (Dasgupta and Sen 1999). A phenomenal extent of production losses of betel vine due to foot rot noted in Bangladesh than any other diseases. (Tanjila et al. 2022).

Farmers face huge economic losses each year due to foot rot. Sclerotium rolfsii Sacc. (teleomorph Arthelia rolfsii) is a polyphagous soil-borne pervasive facultative saprophyte (Paul et al. 2023), with a widespread choice of host. Globally S. rolfsii causes striking crop losses and the disease is generally denoted to as a southern blight (Paparu et al. 2020). Sclerotium rolfsii, commonly known as sclerotium root rot or southern stem rot, was first identified in tomato plants (Solanum lycopersicum L.) in 1892 by Rolfs (Rolfs 1892). This disease primarily occurs in tropical and subtropical regions (Punja 1985). This fungus survives by forming sclerotia in adverse environments and, therefore, is very difficult to control (Mahadevakumar et al. 2015).

Variability of S. rolfsii isolates from different geographical locations was reported by several researchers (Harlton et al. 1995; Okabe et al. 1998; Sarma et al. 2002). The randomly amplified polymorphic DNA (RAPD) might be useful to identifying the polymorphisms in fungi isolates (Welsh and McClelland 1990). RAPD analysis is a powerful tool for the investigation of genetic relatedness and diversity among closely related strains and valuable for differentiating genetic variability of S. rolfsii isolates (Bernardo and Itoiz 2004). The present study has been undertaken to evaluate disease occurrence on betel vine by S. rolfsii, analyze the molecular data by RAPD and ITS rDNA sequencing and morphological characteristics of the pathogen, and test their pathogenicity.

Materials and methods

Survey of foot rot disease of betel vine in northern part of Bangladesh

A survey of the foot rot disease of betel vine was conducted at northern regions of Bangladesh covering districts Rajshahi, Chapainawabganj, and Pabna. During the survey foot rot infected betel vine boroj was monitored, and incidence of disease was recorded. The disease incidence was recorded by Siddaramaiah et al. 1978).

$$\text{Disease incidence}\left(\%\right)=\frac{\text{Total No}.\text{of Infected Plants}}{\text{Total No}.\text{of Plants}}\times 100$$

Isolation of Sclerotium rolfsii from infected betel vine and culture maintenance.

Infected plant parts of the betel vine were collected from different boroj and brought to the Plant Pathology Laboratory for further study. Samples were washed under running tap water, dried in a laminar airflow, and the infected tissue was cut into small pieces (approximately 5 × 5 mm), and surface-sterilized by dipping in 5% Clorox (5.25% sodium hypochlorite) for 3 min. Then the samples were transferred to potato dextrose agar (PDA) supplemented with rifampicin to inhibit bacterial growth and incubated for 7 days at 28 ± 2 °C. Resulting hyphae were transferred to PDA, to establish pure cultures. A total of 22 S. rolfsii isolates were recovered. The isolates were assigned identification numbers (Table 1) and deposited in the culture collection of the laboratory of the Plant Pathology Mycology and Microbiology, Rajshahi University, Bangladesh.

Table 1.

The list of locations and the S. rolfsii isolates

Sl. no	Locationsa	Isolatesb
1	Bagmara	BA-1, BA-2, BA-3, BA-4, BA-5, BA-6
2	Mohanpur	MA-1, MA-2
3	Mougachi	MO-1, MO-2
4	Paba	PO-1, PO-2
5	Durgapur	DU-1, DU-2
6	Puthia	PU-1, PU-2
7	Chapainawabgonj	CH-1, CH-2
8	Pabna	PA-1, PA-2
9	Natore	N-1, N-2

^aList of the surveyed locations

^bS. rolfsii isolates collected from the correspondent surveyed locations

Pathogenicity test of Sclerotium rolfsii

For the pathogenicity test, betel vine cuttings were grown in polybags containing loamy soils. Then the betel vine plantlet was inoculated with a sclerotial suspension of S. rolfsii when two to three seedlings were raised in each polybag. For the preparation of a sclerotial suspension, each isolate of S. rolfsii was grown on PDA plates and 20 ml of double-distilled water was added to the plate following incubation and shaken well to dislodge sclerotia to the plate. A total of 100 ml of sclerotial suspension was prepared for each isolate. The sclerotial suspension was mixed well with soil and betel vine seedlings were transplanted into the inoculated soil. The betel vine seedlings were covered by polythene and incubated at 28 ± 2 °C for 2 days. The inoculated plants were observed every day and watered carefully to keep them under moist conditions After 3–4 weeks the stem of the inoculated plants developed characteristic symptoms similar to that seen in the field. The fungus was reisolated and compared with the original isolates.

Morphological characterization and analysis of morphological diversity

Fungal strains were grown on PDA and incubated at 28 ± 2 °C to examine morphological characteristics. Mycelial discs (6 mm) were cut from the margins of 3-day-old colonies, transferred aseptically to the center of PDA plates, and incubated at 28 ± 2 °C. We observed the mycelial growth (mm) after different incubation periods, growth rate (mm/day), evaluated the number of sclerotia per plate and measured the sclerotial size, shape, weight, and color for each of the strains using a microscope (Model: ML2600, Meiji Techno, Saitama, Japan) with an ocular micrometer with a digital camera. The cultural characteristics and sclerotial characters of S. rolfsii were compared with the chart shown in Supplementary Table 1 (Banakar et al. 2017).

To study the morphological diversity among the isolated S. rolfsii strains, mycelial discs (6 mm) were cut from the margins of 3-day-old developing colonies, placed aseptically on PDA plates, and incubated at 28 ± 2 °C for 25 days. Three replications were used for each isolate. Morphological characteristics of all the isolates were recorded as mentioned above. The isolates were grouped with the maximum similarities based on the UPGMA (Unweighted Pair-Group Method on Arithmetic mean) analysis by MVSP (Multi-Variable Statistical Package) program (version 3.2).

Testing the somatic compatibility of the S. rolfsii isolates

To test somatic compatibility among the isolates, 6 mm mycelial discs were taken from a 4-day-old culture and placed on 85 mm diameter petri plates that were approximately 2 cm apart from each other. All plates were incubated at 28 ± 2 °C in the dark for 10 days. The experiment included three replications and was repeated twice. Isolates that grew together and failed to show a barrage reaction at the colony junction were classified into the same somatic compatibility groups (SCGs), while isolates showing a barrage zone were classified into different SCGs.

DNA extraction

The total genomic DNA from the isolated fungi was extracted by the method described by Raeder and Broda (1985) with a slight modification. All 22 isolates of S. rolfsii were cultured on PDA. In a 250-ml conical flask containing 100 ml of potato dextrose broth (PDB), the hyphal tips from four-day-old pure cultures were inoculated and placed on a shaker for four days at 120 rpm at 25 °C. Then the mycelial mats were filtered through filter paper, blotted to dryness, folded into an aluminum foil, and then preserved at  – 20 °C. Frozen mycelia were ground into fine powder using liquid nitrogen under aseptic conditions. Powdered mycelium (50 mg) was poured into a 1.5 ml Eppendorf tube and homogenized with 500 µl of extraction buffer (250 mM NaCl, 200 mM Tris–HCl pH 8.5, 100 mM EDTA) (Sigma-Aldrich® Brand, Merck, Tokyo, Japan) by vortex. A little over 100 µl of 10% sodium dodecyl sulfate (SDS) was added to an Eppendorf tube that already had the powdered mycelium and extraction buffer in it. The tube was then turned upside down five or six times and left to sit at 65 °C for 30 min to mix. It was then mixed homogenously with 350 µl of phenol and 150 µl of chloroform and centrifuged (13,000 rpm) for 30 min. The upper aqueous phase was then transferred to new tubes without touching the interface. It was mixed with two-thirds of an aliquot of isopropanol and kept at  – 20 °C for thirty minutes. After that, it was centrifuged at 13,000 rpm for ten minutes. To precipitate the DNA, two volumes of absolute ethanol (99.5%) were added to the DNA suspension and kept on ice for 10 min to allow the DNA to precipitate. After precipitation, the mixture was centrifuged at 13,000 rpm at 4 °C for 5 min. Then pour off the supernatant gently and wash the pallet with 70% ethanol. The Eppendorf tube containing the DNA pellet was dried in a vacuum desiccator for two minutes. The pellet was re-suspended in 50 µl of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA, Sigma-Aldrich). The DNA solution was preserved at  – 20 °C for further studies. Before conducting the PCR, the DNA was checked by running it on a 2% agarose gel and quantified with a micro-spectrophotometer (K2 800 nucleic acid analyzer, China).

PCR amplification

For the RAPD test, four primers were used and the primer names and sequences are listed in Supplementary Table 2. For PCR amplification, a total of 20µl of the reaction mixture was prepared including 10µl of 10X PCR Master Mix (Promega, USA), 1 ng of fungal genomic DNA, 1µl of 10 pmol of RAPD primer, and brought up to 20 µl DEPC water. The PCR was as follows: initial denaturation at 94 °C for 2 min and then denaturation at 94 °C for 15 s, annealing at 29 °C for 30 s, extension at 72 °C for 1 min, and these steps are repeated for 35 cycles, and then final extension at 72 °C for 5 min and hold at 4 °C.

Gel electrophoresis

The PCR product (10µl) with 2µl of 6X loading dye was loaded into the well of an agarose gel and run for 25 min at 100 V. The gel was then removed from the gel box and stained with ethidium bromide solution (0.5 µl) for an hour. The stained gel was rinsed with water and photographed with a gel documentation (ChemiDoc MP Imaging System, Bio-Rad, California, USA) for measuring the bands of amplified DNA fragments.

PCR amplification and rDNA sequencing

PCR was performed using a Thermocycler (peqSTAR, Peqlab, Germany). The universal primers for PCR were obtained from Invent Technology, Bangladesh. The primer pairs ITS5-5′-CGGATCTCTTGGTTCTGGCA-3′ and ITS4-5′GACGCTCGAACAGGCATGCC-3′ were used for rDNA amplification. The PCR amplification was carried out in a 25 µl reaction mixture containing 1 ng of DNA sample, 5 µl of 5X PCR buffer, 2.5 mM MgCl₂, 2.0 µl of 2 mM dNTPs (Promega, USA), 20 pmol of each forward and reverse primer (1.0 µl), and 0.2 µl of Taq DNA polymerase and made up to 25 µl with nuclease-free water. The PCR conditions include initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, followed by primer extension for 30 s at 72 °C, and final extension at 72 °C for 10 min. The amplicon was gel purified by PCR clean-up kits (Promega, USA) and sent for sequencing to Aplical Scientific Sdn Bhd, Selangor, Malaysia. Based on the morphological characteristics of the fungi, six isolates were sequenced namely DU-1, BA-1, BA3, CH-1, CH-2, and MA-1.

Sequence analysis

The assembly of the fungal genome was performed using the web service of the NCBI Multiple Sequence Alignment Viewer 1.24.1 (https://www.ncbi.nlm.nih.gov/projects/msaviewer/). ITS-rDNA sequences were aligned using the CLUSTALW program (Larkin et al. 2007). The assembled sequenced contain partial sequences of 18S ribosomal RNA gene, ITS-1, 5.8S ribosomal RNA gene, ITS2 complete sequence, and partial sequence of 28S ribosomal RNA partial sequence and these sequences were submitted to NCBI. The accession numbers of these S. rolfsii isolates are listed below: MH513999 (DU-1), MH514000 (BA-1), MH514001 (BA-3), MH514002 (CH-1), MH514003 (CH-2), and MH514004 (MA-1). A phylogenetic tree was constructed based on the Maximum Likelihood method using MEGA11 (Tamura et al. 2021) by means of the Tamura 3-parameter model (Tamura 1992) with 500 bootstrap replications. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 1.8313)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Statistical analysis

Data were subjected to analysis of variance (ANOVA) and the treatment means were compared using Least Significant Difference (LSD) and Duncan’s multiple range test (DMRT) (p ≤ 0.05) with the SPSS program (version 15). Combined morphological data of twenty-two characters were analyzed with the Unweighted Pair-Group Method on Arithmetic mean (UPGMA) by MVSP (Multivariable Statistical Package). The “corrplot” function in the ggplot2 package of the R programming software (version 4.3.1) was used to do the cluster analysis. The purpose of implementing a cluster analysis was to group various data points so that there is a high degree of relationship between the data sets if they are in the same group and a low degree of correlation if they are in separate groups. The ggplot2 package uses both the complete hierarchical clustering approach and the euclidean distance method for data clustering.

Results

Survey on foot rot disease incidence

The incidence of foot rot on betel vine was recorded in northern Bangladesh from June 2011 to December 2013. In total, 22 isolates were collected from nine different locations (Table 1). In the 2011–2012 cropping season, it was evident that among the nine locations, the highest disease incidence was observed in Mohanpur (51.90%), followed by Durgapur (49.45%), and the lowest was in Chapainawabganj (28.31%) (Table 2). On the contrary, in the 2012–13 cropping season, the highest disease incidence was recorded in Bagmara (54.09%), followed by Durgapur (52.68%), and again, the lowest occurrence of the disease was observed in Chapainawabganj (18.89%) (Table 2). Among the nine locations, the percent disease incidence increased in five locations, namely Bagmara, Durgapur, Puthia, Natore, and Pabna, and decreased in the remaining four (Table 2). Based on the data shown in Table 2, a principle component analysis was done. We discovered six distinct groups based on the incidence of betel vine foot rot disease in nine northern upazilas of Bangladesh (Fig. 1). The disease’s occurrence maintained an identical trajectory in Pubna, Durgapur, and Mohanpur, as well as Bagmara and Mougachi upazilas (Fig. 1). The other four locations formed significantly different clusters. As a consequence, it proved that the betel vine foot rot disease occurrence pattern in various locations differed significantly. Moreover, among the 22 S. rolfsii isolates, the highest disease incidence was found in the case of the isolate BA-1 (28.32%), followed by the isolate BA-2 (22.00%), while the lowest disease incidence was found in the case of the isolates PA-1 and PA-2 (4.00%) (Fig. 2D).

Table 2.

The incidence of foot rot disease of betel vine recorded in Northern Parts of Bangladesh during June 2011 to December 2013

S n	L	TNB	TNP		TNIP		DI
			2011–12	2012–13	2011–12	2012–2013	2011–12	2012–13
1	Bagmara	19	8151	13,664	7525	7392	46.50	54.09
2	Mohanpur	15	3733	5431	2535	4231	51.90	49.20
3	Mougachi	20	9222	11,253	8069	10,433	45.11	40.33
4	Poba	9	6759	5529	2533	1527	37.47	27.61
5	Durgapur	10	3094	5983	1537	3152	49.45	52.68
6	Puthia	10	5537	8932	2575	4275	44.50	47.86
7	Chapainawabganj	11	2736	2736	1869	517	28.31	18.89
8	Natore	10	12,672	11,503	6575	4229	36.76	41.88
9	Pabna	8	2736	6064	1117	1921	31.67	40.82

[Key: L  Location, TNB  Total No. of boroj, TNP  Total No. of plant, TNIP  Total No. of infected plants, DI  Disease incidence.]

Fig. 1.

Principle component analysis (PCA) of the disease incidence of the betel vine foot rot disease. Data presented in Table 2 are utilized for conducting PCA analysis

Fig. 2.

A Healthy and infected betel vine plants, B Healthy and infected stem of the betel vine plants, C Sclerotium rolfsii pure culture on PDA, D Foot rot disease incidence (%) of the twenty-two S. rolfsii isolates

Identification of the virulent isolate/s of the pathogenic fungi

The S. rolfsii isolates were inoculated on betel vines to assess their pathogenicity, and the symptoms of foot rot appeared after 30 days. S. rolfsii infected the betel vine stem near the soil level. At first, the plants turned yellow and finally dried out to a pale brown color. The infected portion of the plants was collected (Fig. 2B), and the pathogens were re-isolated. The pathogen showed the same traits on the PDA medium (Fig. 2C) as it did when it was first isolated from betel vine plants with natural foot rot (Fig. 2A).

Mycelial growth of S. rolfsii isolates

Significant (p ≤ 0.05) variation was observed in mycelial growth of S. rolfsii isolates from betel vine (Table 3). The isolates were divided into three distinct groups according to growth rate: slow, fast and medium. S. rolfsii isolates that had a growth rate of 0.9–1.37 mm per day with an average of 1.14 mm/day were grouped as slow-growing. Comparatively fast-growing isolates had rates ranging from 1.81 to 1.84 mm/day with an average of 1.83 mm/day (Table 3). According to growth rate, slow-growing isolates were DU-1, MA-2, MO-2, PO-1, PO-2, PU-2, N-2; medium growing isolates were BA-2, BA-3, BA-4, BA-5, BA-6, CH-1, DU-2, MA-1, MO-1, N-1, PA-2, PU-1 and fast-growing isolates were BA-1, CH-2 and PA-1. The highest growth rate (1.84 mm/day) occurred with the BA-1 isolate collected from Bagmara Upazilla. In contrast, the DU-1 isolate from Durgapur Upazila showed the lowest growth rate (0.9 mm/day). The mycelial growth rate rapidly increased up to 2 days, then slightly decreased until culture plates were filled (Table 3). Therefore, from these data, we concluded that S. rolfsii isolates collected from various locations showed significant variation in their mean mycelia growth rate.

Table 3.

Mycelial growth of different S. rolfsii isolates on PDA plates during 5 days of incubation

Isolates	Mycelial growth (cm) after different incubation periods (days)				Growth rate (mm/day)	Type of the growth
	2nd	3rd	4th	5th
BA-1	17.5f−I	36.5 h	79f	90d	1.84 k	Fast
BA-2	20.5c−f	46.75 cd	68.5d	87bc	1.74 h	Medium
BA-3	35.5a	65.25a	83.75a	85.3c	1.71 h	Medium
BA-4	28.5b	54.25b	72.5c	86.3bc	1.73 h	Medium
BA-5	10j	18 m	41hi	76d	1.52 g	Medium
BA-6	16d−I	24gh	50.5e	76.5a	1.53 g	Medium
CH-1	10j	34 h	65d	89.5ab	1.79i	Medium
CH-2	3.75a	57.25b	73.8c	80ab	1.81j	Fast
DU-1	14.5hi	23.5li	40i	45i	0.9a	Slow
DU-2	18c−h	30.25j	47.5 fg	70.3ef	1.41f	Medium
MA-1	17.75efghi	41.5f	65.75d	89.3abc	1.79i	Medium
MA-2	14.5ghi	28.5j	44.25d	64.5 h	1.29d	Slow
MO-1	20.0cdef	39.5j	59.25e	72e	1.44f	Medium
MO-2	10j	20.5 m	35j	62 h	1.24b	Slow
N-1	17.25e−I	35.25 h	57.5e	76.8d	1.54 g	Medium
N-2	19.75c−g	33i	47.5 fg	61 h	1.22b	Slow
PA-1	22.5c	47.75c	77.5b	90ab	1.81j	Fast
PA-2	26b	36.25 h	60e	86.75bc	1.74 h	Medium
PO-1	15.5ghi	28.5j	45gh	68.3 fg	1.37e	Slow
PO-2	13.25ij	28.75j	50f	68.5f	1.37e	Slow
PU-1	21.75 cd	43.75dc	67.0d	87.5bc	1.75i	Medium
PU-2	21cde	35 h	45gh	62.5 h	1.25c	Slow
± SE	0.9	1.44	1.64	1.56	0.03
LSD (P ≤ 0.05)	1.23	2.702	2.4	2.61	0.8

Values in a column having the same letter do not differ significantly (p ≤ 0.05) according to DMRT

Morphological diversity among the isolates of S. rolfsii

Morphological variation among the twenty-two isolates of S. rolfsii was studied after 25 days of incubation. The isolates showed a high degree of morphological variation (Table 4, and Figs. 3, 4). Some isolates produced a moderate quantity of aerial mycelium on the colony surface as well as on the lid (Supplementary Fig. 1). Irrespective of all isolates, mycelia were hyaline and produced white to brown sclerotia on the surface of the PDA (Table 4). The number of sclerotia/plates varied from 55 to 950 among the isolates (Table 4). The maximum number of sclerotia (950) was found in isolate BA-1, followed by isolates PA-1 and CH-2, where 830 and 800 sclerotia, respectively (Table 4). The lowest number of sclerotia (55) was found in the MA-2 isolate. However, the diameter of sclerotia ranged from 0.91 mm to 2.0 mm. The maximum diameter of sclerotia (2.0 mm) occurred with the MA-2 isolate and the minimum diameter of sclerotia (0.91 mm) occurred with the N-2 isolate (Table 4).

Table 4.

Color, number, diameter, and weight of sclerotia of different isolates of S. rolfsii on PDA plates after 25 days of incubation

Isolate Code	Color of sclerotia	No. of sclerotia/plate	Diameter (mm) of sclerotia	Weight of per 100 sclerotia (mg)
BA-1	B., W	950a	1.30b	6a
BA-2	B, W	280d	1.09e	2d
BA-3	W, O. W, B	120f	1.13d	4c
BA-4	B, O. W, W	110f	1.09d	3c
BA-5	W,B	270d	1.36b	7a
BA-6	L. B, D. B	180e	1.03e	5b
CH-1	O. W, B	350c	1.03e	1e
CH-2	O.W	800a	1.47b	1e
DU-1	B	120f	1.31b	3c
DU-2	B	200d	1.31b	3c
MA-1	B, O.W, W	160e	1.17d	5b
MA-2	W, B	55 g	2.0a	4c
MO-1	B	500v	1.11d	3c
MO-2	W,B,O. W	240d	1.03e	1e
N-1	O.W,B	270d	1.15c	2d
N-2	L. B	630b	0.91f	4c
PA-1	O. W	830a	1.21c	1e
PA-2	O.W, W, B	150e	1.25b	2d
PO-1	B, W	400c	1.25b	2d
PO-2	B, O.W, W	65 g	1.09e	5b
PU-1	W, L. B, B	340d	1.31b	1e
PU-2	D. B, O. W, W	275d	1.18c	2d

Here, W  White, O.W  Off white, B  Brown, L.B  Light brown, D.B  Dark Brown. Values in a column having the same letter do not differ significantly (p ≤ 0.05) according to DMRT

Fig. 3.

Pattern of the sclerotia production of the twenty-two isolates of the S. rolfsii on PDA after 25 days of incubation. Based on the sclerotia production the isolates were grouped into six different groups. The left panel showed the six distinct group of isolates and the pattern of sclerotia production by S. rolfsii isolates on PDA

Fig. 4.

The size and shape of the sclerotia of the twenty-two isolates on PDA after 25 days of incubation

The growth and distribution pattern, size, and shapes of the sclerotia indicated that the variation existed among the isolate (Figs. 3, 4). The color of sclerotia varied from white to dark brown (Figs. 3, 4, and Table 4). Six types of sclerotial growth patterns were observed—(i) sclerotia produced near the point of inoculation [PA-2, CH-2, MA-1, PU-1, DU-2, NA-2, MO-1, MA-1, BA-3, MO-2, CH-1, BA-1]; (ii) sclerotia scattered all over the plate [DU-1]; (iii) aggregated sclerotia on the zonal area [N-1, PA-1, BA-4]; (iv) sclerotia grown on the embedded parts of the mycelium [N-2, BA-2]; (v) sclerotia grown peripherally [PO-1, PU-2, MA-2]; and (vi) sclerotia grown on the aerial parts of the mycelium [PO-2] (Fig. 3).

Analysis of morphological diversity of S. rolfsii isolates with UPGMA

Combined morphological data the isolates were analyzed with UPGMA by MVSP and the results showed six groups at a 62% similarity level (Fig. 5). Twelve isolates were clustered in Group 1, which further constituted three sub-groups at a 75% similarity level. Group 2 consisted of 1 isolate at a 65% similarity level. Group 3 contained 3 isolates at a 70% similarity level. Group 4 contained 2 isolates at a 73% similarity level. Group 5 contained 3 isolates at 79% similarity level and Group 6 contained 3 isolates at a 79% similarity (Fig. 5). Group 1 and Group 3 grew more slowly than Group 2 and Group 4. Group 4 consisted of two isolates i.e. N-2 and BA-2 (Fig. 5), containing light brown sclerotia and abundant fluffy type mycelia which was characteristically different from other cluster groups.

Fig. 5.

Analysis of the combined morphological data of twenty-two isolates with UPGMA by MVSP and the S. rolfsii isolates was grouped into six distinct clusters. The dendrogram shows six different groups by color

The link between betel vine foot rot disease incidence, S. rolfsii mycelial growth rate, and different sclerotial characters are shown in Fig. 6 and Supplementary Table 4. The correlation study was carried out using the data provided in Supplementary Table 3. Positive correlation is shown in “Blue” color and the negative correlation is shown in “Red” color (Fig. 6). Mycelial growth rate has shown 40% positive correlation with the disease incidence (Fig. 6).

Fig. 6.

Correlation plot showing the relation between the betel vine foot rot disease incidence and S. rolfsii mycelial growth rate and others morphological characters

Somatic compatibility test

After co-culturing the isolates, it was observed that in the case of an incompatible interaction, mycelial lysis occurred and the development of a clear barrage zone (B) was observed at the mycelial contact region (Fig. 7A and B). With incompatible isolates no barrage (NB) zones developed (Fig. 7C and D). A total of 122 combinations were conducted with the 22 isolates; 38 combinations were somatically compatible and the remaining 84 were incompatible (Data not shown).

Fig. 7.

Somatic compatibility test by co-culturing different S. rolfsii isolates on PDA plates. A total of 122 combinations were tested by the twenty-two isolates and found 38 combinations were found somatic compatible with clear barrage zone (A–B) and 84 were somatic incompatible with no barrage zone (C–D). B indicates on the PDA plates barrage zone and NB indicates no barrage zone respectively

Molecular characterizations of S. rolfsii

DNA fingerprint analysis by RAPD and ITS region amplification by PCR

Genetic variation was occurred among the twenty-two isolates using RAPD primers (Supplementary Table 2). Only the RAPD-4 primer produced distinct and clear bands. No amplification occurred with the other three primers (RAPD-1, RAPD-2, RAPD-3) (Fig. 8A). In this study, it is indicated that the RAPD-4 primer showed a hundred percent polymorphism. The amplicon size 750 bp of the ITS region was amplified using ITS4 and ITS5 primers which include the partial sequence of 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 complete sequence, and partial sequence of 28S rRNA sequence Fig (Fig. 8B&C).

Fig. 8.

Gel electrophoresis of RAPD fragments obtained with RAPD primer-4 (A) and PCR amplification of the ITS region with ITS-4 and ITS- 5 Primers (B & C). Lane 1–22 are the S. rolfsii isolates BA-1, BA-2,BA-3,BA-4,BA-5,BA-6,MA-1,MA-2, MO-1,MO-2, PO-1,PO-2, DU-1,DU-2,PU-1,PU-2,CH-1,CH-2,N-1,N-2,PA-1,PA-2 respectively. L indicates the DNA marker

Multiple sequence alignment and phylogenetic analysis

The ITS (internal transcribed spacer 1) region of the six selected S. rolfsii isolates (BA-1, BA-3, CH-1, CH-2, DU-1, and MA-1) was sequenced and all sequences showed 98–100% similarity with sequences of Athelia rolfsii (anamorph: S. rolfsii) from GenBank by nBLAST search analysis. Figure 9 shows the maximum-likelihood tree created using the sequence data of the six selected S. rolfsii isolates and the reference sequences from the NCBI database. Rhizoctonia solani was included as an outgroup member. This analysis involved 35 nucleotide sequences. There were a total of 1732 positions in the final dataset.

Fig. 9.

Evolutionary analysis by Maximum Likelihood method. The evolutionary history was inferred using the Maximum Likelihood method and Tamura 3-parameter model. The tree with the highest log likelihood ( – 6531.27) is shown. This analysis involved 45 nucleotide sequences. There were a total of 1730 positions in the final dataset. Evolutionary analyses were conducted in MEGA11

The results of the multiple sequence alignments are shown in Fig. 10. The partial sequence of the 18S rRNA gene was almost identical in the six selected isolates. While the isolates CH-1 and CH-2 had 39 mismatches with the previously sequenced isolates, the isolates BA-1, BA-2, and DU-1 had 20 mismatches, whereas the isolate MA-1 contained 32 mismatches (Fig. 10).

Fig. 10.

The results of the multiple sequence alignment betel vine isolates from Bangladesh and other related isolates of S rolfsii retrieved from NCBI. The asterisk indicates the betel vine isolates used in this study

Discussion

Sclerotium rolfsii Sacc. is an aggressive, soil-borne fungi. This pathogen is globally dispersed and ubiquitous in nature (Wang et al. 2023). Numerous studies have documented substantial phenotypic variability among S. rolsfii isolates from various species and geographically distinct locales (Sarma et al. 2002; Remesal et al. 2012; Xie et al. 2014). Due to the pathogen’s widespread occurrence, broad spectrum, and capacity to cause persistent sclerotia, it is challenging to treat (Remesal et al. 2012; Xie et al. 2014; Jebaraj et al. 2017). Moreover, during pathogenesis, S. rolfsii produces oxalic acid (Monazzah et al. 2018) and various cell wall degrading enzymes (CWDEs) (Kishore et al. 2005). The synergistic effects of the fungal oxalic acid and these CWDEs degrade the host cell walls and enable the pathogen to cause severe infection (Prova et al. 2018; Karim et al. 2019). Therefore. understanding the biology and genetic diversity of S. rolfsii is crucial for developing effective management methods (Khatri et al. 2017). In this present study, a total of 22 isolates were isolated from the betel vine in northern part of Bangladesh. The isolates were characterized based on their morphological and molecular characteristics. Morphological diversity was observed in mycelial growth rate, number, shape, size, and color of sclerotia. Sclerotium rolfsii form sclerotia as a survival structure which serves as the primary inoculum. Sclerotia are an extremely hardy and relatively resistant survival structures (Paparu et al. 2020). Singh and colleagues reported that profuse mycelial growth and sclerotial production contributed to the considerable crop losses associated with S. rolfsii (Singh et al. 2003). Since the S. rolfsii basidial stage is rarely observed, this fungus is characterized by the morphology of sclerotia in most cases (Muthukumar and Muthukumar 2013).

In an earlier study reported that Sclerotium wilt of betel vine in thirty selected gardens in Chittagong division of Bangladesh (Mridha and Alamgir 1989). Our survey in 122 gardens (boroj) in four northern districts revealed that Bagmara had the highest incidence of foot rot disease (54.09%) and Chapinawabganj had the lowest (18.89%). During a survey conducted by Masud et al. 2020 at five different upazillas in five districts of Bangladesh, the highest foot and root rot incidence (27.80%) was found in August at Gouranadi upazilla, and the lowest (6.00%) was at Sitakundo upazilla. Jahan and colleagues investigated foot and root rot disease of betel vine (Piper betle L.) in Kushtia district of Bangladesh and reported the maximum disease incidence was recorded in Mirpur Upazila where disease incidence ranged from 54 to 64% and the minimum disease incidence was recorded in Khoksha Upazila where disease incidence ranged from 28 to 34% (Jahan et al. 2016). The present study also supports these previous findings, and it was noted that foot rot disease incidence is increasing in northern regions of Bangladesh.

The pathogenicity test showed that the BA-1 (Bagmara-1) isolate to be the most virulent (Fig. 1D). Differences in virulence among S. rolfsii isolates have also been reported in previous studies. In that study, the cross inoculation of the isolates of S. rolfsii from groundnut, wheat, potato, guava, and Bengal gram, had identified groundnut as the most susceptible host for S. rolfsii (Sarma et al. 2002). Therefore, variability in S. rolfsii virulence can occur both in host and isolate-dependent manner (Jahan et al. 2016).

Our study has confirmed that the isolates of S. rolfsii isolates have wide-ranging variations in the characters such as colony morphology, mycelial growth rate, sclerotia production, sclerotia size, and color. Out of the twenty-two isolates, the highest mycelial growth rate was recorded in the BA-1 isolate and the lowest was in DU-1 (Table 3). It was also observed that the sclerotia of S. rolfsii isolates were mostly round to oval in shape and the number of sclerotia varied from 55 to 950 per plate (Table 4). These variabilities among the S. rolfsii isolates were also reported by some other previous studies. A study conducted on the twenty-six Indian S. rolfsii isolates which were collected from different hosts, identified variability in growth rate and basidial stage production (Sarma et al. 2002). Another research group had found the average sclerotial diameter of 1.0 ± 0.2 mm at 20 °C and five types of sclerotial color, while most of the sclerotia were dark brown (Punja and Damiani 1996).

Morphological and genetic variability among S. rolfsii populations were also reported where out of 17 isolates, most of the isolates had compact colonies and few had fluffy colonies; furthermore, based on the growth rate, S. rolfsii isolates were categorized into three groups—slow-growing, fast-growing and intermediate (Prasad et al. 2010). Similarly, in this present study, it was also observed that twenty-two isolates were categorized into six groups according to the growth rate (Fig. 5), and compact and fluffy colonies were also observed (Supplementary Fig. 1). These findings were consistent with the earlier investigations.

It was evident that the formation sclerotia by S. rolfsii depend on many factors such as nutritional and non-nutritional factors, nutrient depletion, constraint of growth by a physical barrier (Punja 1985). However, we have found that the sclerotia of some isolates showed a shiny appearance due to the presence of gummy material on their surface (Figs. 2A and 3B). The presence of gummy material due to the production of extracellular polysaccharides by these isolates. Filamentous fungi are a very promising producer of 1, 3 D-glucan as the hyphal cell wall and extracellular matrix contain more than 75% polysaccharides (Flieger et al. 2003).

The combined morphological data of twenty-two characters were analyzed with UPGMA by MVSP and the results showed six groups at a 62% similarity level (Fig. 5). The UPGMA method is a straightforward approach for constructing a dendogram from a distance matrix (Sokal and Michener 1958) and has been used frequently for the morphological characterization of filamentous fungi (Abbasi et al. 2016).

Somatic compatibility and/or incompatibility test was conducted within 22 isolates of S. rolfsii. Among the twenty-two isolates, seven pairs of the isolate showed mycelial compatibility, and fifteen showed mycelial incompatibility (Fig. 3A). Mycelial compatibility and incompatibility interaction by different species was also reported in previous studies. Punja and Sun (2001) evaluated genetic diversity among mycelial compatibility groups of S. rolfsii (teleomorph: Athelia rolfsii) and S. delphinii. It has been reported that when mycelia of different isolates belonging to the same species confront one another, on a suitable growth substrate, a distinct zone of demarcation (barrage or aversion zone) was developed between the colonies (Papaioannou et al. 2015). Recognition of non-self from self is the underlying basis of the incompatible reaction (Punja and Sun 2001). Mycelial compatibility reaction was also used by Sarma and colleagues to study the variability and relatedness among fungal species belonging to different geographical regions (Sarma et al. 2002).

For the molecular characterization of the selected S. rolfsii isolates RAPD-PCR technique was utilized and genetic variability was confirmed among the Bangladeshi S. rolfsii isolates. Genetic variability among the isolates of S. rolfsii was studied by many researchers using molecular techniques like RAPD, ITS-PCR, and RFLP. Shokes and colleagues tested the pathogenic variability and pathogenic potential of S. rolfsii isolates on the groundnut variety TGCS888. The molecular variability among the isolates of S. rolfsii was studied using the ITS region of rDNA, Random Amplified Polymorphic DNA (RAPD), and Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP) (Shokes et al. 1996). In their study, Okabe and colleagues divided 67 isolates of the southern blight fungus from Japan into five groups based on ITS-RFLP analysis of nuclear rDNA—three groups were reidentified as S. rolfsii and two resembled S. delphinii in RFLP patterns (Okabe et al. 1998). The present results also support the above findings.

Phylogenetic analysis of all ITS sequences of the isolates from the present study revealed that the references sequences of A. rolfsii clustered together in a group and suggested that this species is closely related to each other (Fig. 5A). S. delphinii and S. coffeicola produced a separate subgroup in the same cluster, which separated these two species from S. rolfsii (Fig. 5A). Similar ITS phylogeny was described earlier on S. rolfsii and supports the findings of the present study (Paul et al. 2017). Harlton and colleagues described that S. rolfsii and S. delphinii clustered together and S. coffeicola separated by different closely related clusters (Harlton et al. 1995). Hence, all the isolates obtained in the present study were identified as S. rolfsii based on the ITS sequence analysis.

Based on the phylogenetic relationship and sequence alignment it is clear that there are variations that exist among the S. rolfsii isolates in Bangladesh. From the phylogenetic analysis, the isolates CH-1 and CH-2 yielded the same group in the same cluster (Fig. 9). These two isolates were collected from the same area but away from the other isolates. Therefore, the microclimates of different regions may influence the pathogen morphology and genetic diversity. Therefore, it is urgent dement to know the genetic variability of S. rolfsii by sequencing other isolates from different hosts and agro-ecological zones in Bangladesh

Supplementary Information

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Data Availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Declarations

Conflict of interest

The authors declare they have no financial or conflict of interest.

Research involving human participants and/or animals

The research accomplished in the manuscript does not involve any human participants or animal preparations.