Plant Pathol J > Volume 38(4); 2022 > Article
Gnanesh, Arunakumar, Tejaswi, Supriya, Manojkumar, and Devi: Characterization and Pathogenicity of Lasiodiplodia theobromae Causing Black Root Rot and Identification of Novel Sources of Resistance in Mulberry Collections

Abstract

Black root rot (BRR) caused by Lasiodiplodia theobromae is an alarming disease of mulberry that causes tremendous economic losses to sericulture farmers in India and China. Successful control of this disease can be attained by screening germplasm and identifying resistant sources. Seventy four diseased root samples were collected from farmer’s fields belonging to four major mulberry growing states of South India. Based on morpho-cultural and scanning electron microscopy studies, 57 fungal isolates were characterized and identified as L. theobromae. Phylogenetic analysis of concatenated internal transcribed spacer and β-tubulin sequences revealed variation of the representative 20 isolates of L. theobromae. Following the root dip method of inoculation, pathogenicity studies on susceptible mulberry genotypes (Victory-1 and Thailand male) recognized the virulent isolate MRR-142. Accordingly, MRR-142 isolate was used to evaluate resistance on a set of 45 diverse mulberry accessions. In the repeated experiments, the mulberry accession ME-0168 which is an Indonesian origin belonging to Morus latifolia was found to be highly resistant consistently against BRR. Eight accessions (G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489, and MI-0501) were found to be resistant. These promising resistant resources may be exploited in mulberry breeding for developing BRR resistant varieties and to develop mapping populations which successively helps in the identification of molecular markers associated with BRR.

Mulberry (Morus spp.) is a perennial fast growing woody plant, which is widely cultivated in many tropical and subtropical areas. The leaves of the mulberry are economically important as they are the only source of feed for the monophagous silkworm (Bombyx mori L.). After China, India is one of the leading producers of mulberry with a total acreage of around 239,926 hectares. Karnataka, Andhra Pradesh, West Bengal and Tamil Nadu are the major silk producing states in India (Central Silk Board, 2020). The tropical climate of South India is highly favorable for mulberry cultivation where the crop is harvested at every 70 days (Gnanesh et al., 2021). Mulberry production is limited by several diseases as the perennial nature of the mulberry provides scope for long-term survival and multiplication of soil-borne pathogens (Sharma et al., 2003). Among the soil-borne diseases, root rot is epidemic in nature and a major limiting factor in the cultivation of mulberry. Root rot causes 30% mortality of plants with a 14% decrease in leaf yield, besides deteriorating the leaf quality (Chowdary, 2006; Rajeswari and Angappan, 2018).
In mulberry, different kinds of root rot have been reported, such as black root rot, dry root rot, charcoal root rot, violet root rot, white root rot, Armillaria root rot, Rhizopus rot, and bacterial root rot (Gnanesh et al., 2021; Radhakrishnan et al., 1995; Sharma et al., 2003; Yoshida et al., 2001). Amongst them, black root rot (BRR) caused by Lasiodiplodia theobromae (syn. Botryodiplodia theobromae), dry root rot (Fusarium solani and F. oxysporum), and charcoal root rot (Macrophomina phaseolina) are frequently reported in India (Pinto et al., 2018).
Lasiodiplodia species are harmful fungal pathogens and have been associated with nearly 500 plant hosts causing various degrees of crop losses. It causes root rot disease in mulberry that affects all parts of the plant and it spreads rapidly affecting a large number of plants in a short period leading to the abandonment of mulberry gardens (Pappachan et al., 2020). The fungus prefers 25-30°C for good growth and survives on all types of substrates. After pruning, the pathogens enter the host through the cut ends of the stem. Once, the plant becomes vulnerable to infection, the fungus dominates inside the roots by multiplying the hyphae rapidly in the cortical tissues and extending up to the pith region. It enters the xylem vessels and causes the death of the plants (Sharma et al., 2003). Diseased plants appeared stunted with dark colored stems and chlorotic leaves, gradually the leaves get wilted and defoliated prematurely. The fine feeder roots and the main root system of these infected plants will damage severely with typical symptoms like discoloration of root xylem followed by the decay of root cortex. Also, the bark of the roots becomes fragile and smelled emanating a foul odour. Gradually as the damaged roots could not firmly hold the plant in the soil they can be easily uprooted (Sowmya et al., 2018; Xie et al., 2014).
For the first time in China, BRR caused by L. theobromae was reported by Xie et al. (2014) and it has a serious impact on the sustainable growth of the local sericulture industry (Xie et al., 2016). Similarly, Sowmya et al. (2018) studied the severity of the BRR disease in major mulberry growing areas of India. The Lasiodiplodia species is known to cause dieback, cankers, and stem and root rot in multiple crops like almonds, blueberry, castor, Chinese hackberry, citrus, cocoa, coconut, grapevines, groundnut, jackfruit, mango, melon, olive, Pinus spp., strawberry, and rice (Bautista-Cruz et al., 2019; Chen et al., 2021; Liang et al., 2019; Nam et al., 2016; Pečenka et al., 2021; Rosado et al., 2016; Saeed et al., 2017).
As it is not sufficient to classify Lasiodiplodia species based on morphological features, DNA sequence-based approaches have been widely recommended (Bautista-Cruz et al., 2019). For many years, L. theobromae was treated as a monotypic genus within the Botryosphaeriaceae (Slippers et al., 2013). However, phylogenetic analysis evidenced the existence of many additional species (de Silva et al., 2019).
Various chemical and biological methods were recommended to manage the disease caused by Lasiodiplodia spp. and avoid economic losses (Kamil et al., 2018). The chemical control measures are non-judicious and undesirable due to the buildup of resistance among pathogens (Leroch et al., 2011). They are also unsafe for the environment and human health and toxic to silkworms (Naik et al., 2010). Biocontrol measures were not very effective due to the influence of various factors on their efficacy (Nelson, 2004). Genetic improvement by breeding a resistant variety ensures a cost-effective and environment-friendly system for the control of plant diseases (Arunakumar et al., 2021; Pandey et al., 2021). Identification and breeding of resistant mulberry varieties is the only justifiable option to achieve sustainable mulberry cultivation.
There are limited data on BRR of mulberry in India, and the previous characterization of L. theobromae was based on morphology. Sowmya et al. (2018) used random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSRs) to study the genetic variability, among the ten isolates of L. theobromae causing BRR of mulberry, similarly, Pappachan et al. (2020) characterized only one isolate of L. theobromae. However, these studies could not reveal the molecular phylogeny of isolates and did not attempt to identify resistant sources to L. theobromae. Several research investigations reported new species of Lasiodiplodia (de Silva et al., 2019; Rosado et al., 2016) indicating the extension of its host range highlights the need for prospecting novel Lasiodiplodia species associated with mulberry. Accurate detection of pathogens is very essential for the development of proper management approaches, moreover use of highly pathogenic or aggressive isolates is necessary for inoculation trials for selecting germplasm accessions with broader resistance to diseases (Oliveira et al., 2021). Also, there is an immediate need to identify resistant sources to transfer resistance genes into elite backgrounds of mulberry.
Thus, the objectives of the present study were as follows: (1) to characterize L. theobromae causing BRR of mulberry in India, morphologically, combined with DNA sequence analysis using internal transcribed spacer (ITS) region and β-tubulin, (2) to evaluate the pathogenicity of the selected isolates (3) finally, to identify BRR resistant resources and their utilization in future mulberry breeding programs to develop root rot-resistant varieties.

Materials and Methods

Sample collection and isolation of the fungus

Seventy four diseased root samples of mulberry were collected from farmers field from 2017 to 2019 in high-incidence states (Andhra Pradesh, Karnataka, Tamil Nadu, and Telangana) of South India. The root rot causative fungal pathogen was isolated following the root bit method of isolation. The infected mulberry roots showing typical root rot symptoms were chopped into small bits and surface-sterilized using 5% sodium hypochlorite for 2 min. These root bits were washed with sterile water and dried up on a filter paper (Whatmann No. 1). Dried root bits were placed on Petri plates with sterile potato dextrose agar (PDA; HiMedia, Mumbai, India) under aseptic conditions. The plates with root bits were incubated at 27 ± 1°C for 7 days to obtain fungal growth. Pure cultures were obtained by sub-culturing matured hyphae on 2% water agar plates and stored for further use at 4°C.

Morphological identification of fungal isolates

The morphology of 7-day-old fungal cultures grown on PDA medium incubated at 27°C under dark conditions was examined. Cultural characteristics like colony color, pigmentation, texture, and growth were noted. Conidial morphology was observed using light microscopy and conidial dimensions of 20 selected isolates were determined (Supplementary Table 1). The length and width of 10 mature and immature conidia per isolate were measured using ImageJ software.

Scanning electron microscopy

Glutaraldehyde (2.5%) prepared in 0.2 M cacodylate buffer (pH 7.2) was used for the initial fixation of fungal cultures. The fixed cultures were washed using cacodylate buffer and then by double distilled water dehydrated in ethanol series. Critical drying of the dehydrated samples was carried out by using a Critical Point Dryer (EMS850, Electron Microscopy Sciences, Hatfield, PA, USA). Lastly, samples were coated with gold nanoparticles in Sputter Coater (EMS550, Electron Microscopy Sciences) mounted onto copper stubs using double side sticky tape. Microscopic images were obtained after scanning under JEOL 100 CX ASID-4D scanning electron microscope (JEOL Ltd., Tokyo, Japan) at 20 kV (Arunakumar et al., 2018; Bozzola and Russell, 1992).

Molecular identification of the pathogen DNA isolation and polymerase chain reaction amplification

Pure cultures of 20 fungal isolates were inoculated in 50 ml of sterile potato dextrose broth and incubated for 7 days at 27 ± 1°C, without shaking. The mycelial mats were separated by using sterilized filter paper (Whatmann No.1). DNA extraction was performed, following the method described by Gnanesh et al. (2021).
The representative fungal isolates of mulberry were identified by sequencing ITS region, ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATAT GC-3′) primers (White et al., 1990) and β-tubulin region, βt2a (5′-GGTAACCAAATCGGTGCTG CTT TC-3′) and βt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass and Donaldson, 1995). The polymerase chain reaction (PCR) amplification was performed with a thermocycler (GeneAmp PCR system 9700, Applied Biosystems, Foster City, CA, USA). in a 25 μl reaction mixture consisting of 2 μl of template DNA (40 ng), 12 μl of 2× Ampliqon master mix, 1.5 μl of each primer (10 pM) synthesized from Eurofins, Bangalore, India and 8 μl of nuclease free water. The PCR parameters for ITS gene PCR amplification: initial denaturation at 94°C for 4min followed by 35 amplification cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and final extension at 72°C for 8 min. Similarly, β-tubulin gene PCR amplification was performed in a 25 μl reaction mixture which contained 10 μl of 2× Ampliqon master mix, 2 μl template DNA (40 ng), 11 μl of nuclease free water and 1 μl of each primer (10 pM). The PCR parameters for partial β-tubulin region are initial denaturation at 95°C for 5min, followed by 35 amplification cycles of 94°C for 45 s, 55℃ for 45 s, and 72℃ for 1 min, and final extension at 72°C for 7 min. Amplified products were electrophoresed using 1.5% agarose gel stained with ethidium bromide with 1× TAE running buffer, and visualized in a UV transilluminator. A 100 bp ladder was used as a marker and gels were documented using GelDoc Bio Imaging System (Syngene, Cambridge, UK). The amplified PCR products were purified and sequenced by Sanger sequencing method, Eurofins Pvt., Ltd. (Bengaluru, India).

Phylogenetic analysis

Sequence alignments of ITS and β-tubulin were carried out using the MEGA X software (Kumar et al., 2018). The obtained nucleotide sequences were edited using BioEdit software and submitted to BLAST search analysis to identify the sequence homology in NCBI database. Finally, the sequences were deposited in GenBank and obtained the accession numbers. The phylogenetic analysis of concatenated data was done by selecting the best nucleotide substitution model using Bayesian Information Criterion (BIC) in Mega X software (Kumar et al., 2018). Tamura 3-parameter (T92) model was selected for phylogeny construction and the tree was rooted to Barriopsis fusca as an outgroup. Type strain and reference sequences were obtained from GenBank and the trees were analyzed by the bootstrap method with 1,000 replications.

Pathogenicity test

Pathogenicity tests of 20 representative isolates were performed on two susceptible mulberry accessions, Victory-1 and Thailand Male, following the root dip method under glasshouse conditions (Arunakumar and Gnanesh, unpublished). Four months old saplings grown in raised nursery beds were gently uprooted and dipped in the conidial suspension (1 × 106 spore load per ml load) for 12 h. Control plants were also dipped in sterile distilled water for 12 h. Later, saplings were transplanted into the earthen pots having sterile soil arranged with three replications following a completely randomized design (CRD). The infected plants were watered regularly to maintain sufficient moisture. BRR symptoms, wilting, and rotting percentage were recorded after 120 days of post inoculation. The pathogenicity of the isolates was classified using the wilting and rotting percentage as described in Table 1.

Screening of diverse mulberry germplasm accessions

Forty five mulberry germplasm accessions including exotic and indigenous collections were used for screening. The accessions represent eight different countries including India (33), Indonesia (3), Japan and Thailand (2 accessions each), Philippines, Australia, China and France one accessions each. Among the 45 accessions 17 accessions belonged to collections, eight were open pollinated hybrids, six were from the survey, five accessions were from selections, and four each accession were from cross pollinated hybrids and clonal selection respectively and only one accession was developed. These accessions belonged to six different Morus spp. including a known popular susceptible variety Victory-1, were chosen for identification of resistance against L. theobromae causing BRR of mulberry (Table 2). Four months old saplings of each accession were uprooted gently and inoculated following the root dip method as mentioned above. The inoculated pots with four replications (single plant/replication) were arranged in a CRD. The experiment was conducted under glasshouse conditions with two control plants free from inoculation and repeated twice. The aerial observation of the total number of leaves, shoots (wilted and healthy), and the number of plants dead was recorded at 30 days intervals in each accession. Plants were uprooted after 120 days of post inoculation and observations on weight of the whole root, healthy root and above ground biomass per plant (g) were recorded. These disease indices like wilting and rotting percentages were calculated according to formulae described by Sowmya et al. (2018). The disease severity index and disease reaction were categorized for each accession based on the wilting and rotting percentage using the modified scale as mentioned in Table 1 (Ahmed et al., 1999).

Statistical analysis

The collected data were statistically processed using IBM SPSS Statistics software version 23.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA analysis was performed to study the difference in the pathogenicity of the selected L. theobromae isolates and means of the BRR disease scores were compared using Tukey test (P < 0.05).

Results

Fungal isolation and morphological identification

Symptomatic mulberry plants in the field exhibited varying degrees of BRR symptoms, characterized by yellowing and wilting of leaves (Fig. 1A), browning of the severely infected roots and in some cases appearance of newly regenerated roots were observed (Fig. 1B). The stem of the symptomatic plants was browning of root xylem and rotting of root cortex (Fig. 1C). Fifty seven isolates of L. theobromae were successfully recovered from 74 root rot samples collected from Southern India. The isolated fungi were grown on PDA media at 27°C in dark conditions. The cultures showed initially off-white to grey with fluffy, round to irregular margin, flat aerial mycelia were grown radially and uniformly in all directions and covered the surface of the media (90 mm diameter Petri dishes) within 4-5 days after incubation (Fig. 2A). Initially, the reverse side of the culture showed algae green dot patches and longitudinal striations towards the periphery. After 14-15 days of incubation, the reverse side turned to black color (Fig. 2B) and the upper surface formed dark grey colonies with fluffy aerial mycelium (Fig. 2C). Liquid exudates were produced and became dry after a few days (Fig. 2D).
The virulent isolate MRR-142 is shown in the microscopic pictures in Fig. 3. The mycelia were hyaline and septate, and some of the malformed mycelia were also observed (Fig. 3A and B). Pycnidia were semi-immersed, solitary, globose, papillate, and leaden-black forms after 15-20 days of incubation. Paraphyses were cylindrical, aseptate and hyaline. Conidiogenous cells were characterized as holoblastic, cylindrical and hyaline. Conidia produced after 15-20 days of incubation, immature conidial length measured from 13.34 μm to 7.4 (± 1.24) μm and breadth were ranged from 4.47 μm to 2.28 (± 0.51) μm (Fig. 3C and D). The mature conidial length was found in the range from 25 μm to 17 (± 3.0) μm and breadth was in the range from 14 μm to 7 (± 2.0) μm (Supplementary Table 1). Conidia were initially hyaline, aseptate, ellipsoid to obovoid, unicellular, cinnamon to sepia, dense walled and rounded with longitudinal light striations, then mature conidia form 1-2 septa (Fig. 3E and F). The conidial morphology and size indicated that the isolates were L. theobromae. The fine structure of conidial surfaces and conidia on a conidiogenous cell was observed using scanning electron microscopy (SEM). Conidial surface morphology of L. theobromae, although appeared to be smooth when viewed in a light microscope they are roughened and verruciform when viewed in high magnification in SEM (Fig. 3G-I). The ornamentation type could not be reliably distinguished with light microscopy. Conidia are broadly ovoid to ellipsoidal, presenting both ends broadly rounded or end rounded and the base narrowed or apiculate (Fig. 3H).

Phylogenetic analysis

Twenty L. theobromae isolates were selected for phylogenetic analysis using ITS and β-tubulin regions based on the geographical region, representing the four states of South India. Also, based on virulence and morphological characters of the isolates like colony color, characters of aerial mycelia growth. The amplicon size of ITS and β-tubulin was detected between 500-600 bp and 300-400 bp, respectively (Munirah et al., 2017). Blast search homology of ITS and β-tubulin sequences revealed 98-100% similarity with type strain CBS 164.96 L. theobromae (Supplementary Table 2). GenBank accession numbers were obtained for all the isolates (Table 3). A phylogenetic tree was constructed for all the isolates of L. theobromae and the isolates were grouped into a single clade, representing the reference sequences (CBS111530 and COAD1788) and type strain (CBS 164.96) with a bootstrap value of 83% (Fig. 4). Thus confirming the identity of the isolated pathogen is L. theobromae, whereas all the other Lasiodiplodia species; L. citricola, L. crassispora, L. euphorbicola, L. mahajangana; L. missouriana; L. pseudotheobromae; L. subglobosa formed monophyletic clade individually with respect to their type strain. The closest species near to L. theobromae isolates were L. missouriana and L. viticola, whereas L. crassispora formed a separate cluster. The final pooled dataset of ITS and β-tubulin of 40 nucleotide sequences after alignment consists of 649 positions, 595 were conserved, 54 variable, 31 parsimony-informative, and 23 were singleton.

Pathogenicity test

The pathogenicity test conducted on susceptible genotypes Victory-1 and Thailand Male revealed significant variation (P < 0.05) between the selected 20 isolates (Table 4). The infected plants, showed similar symptoms of BRR, like the appearance of dark colored lesions, rotted roots, wilting of leaves and browning of stems (Fig. 5). The wilting and rotting percentage of Victory-1 ranged between 3.6 to 97.6 and 4.6 to 100.0, respectively, similarly of Thailand male, 7.6 to 99.3 and 10.3 to 95.0, respectively. Sixteen isolates were highly pathogenic and four isolates, MRR-056, MRR-126, MRR-133, and MRR-153 were less pathogenic on both the susceptible genotypes exhibiting low levels of wilting and rotting. Among all the isolates, MRR-142, isolated from Andhra Pradesh, was found to be highly pathogenic which caused a maximum percentage of wilting and rotting (Table 4).

Screening of resistant mulberry genotypes

A total of 45 mulberry accessions were screened against the L. theobromae isolate MRR-142 through the root dip method of inoculation. The BRR disease reactions of resistant and susceptible accessions revealed significant variation under glasshouse conditions. The wilting and rotting percentage among the 45 accessions in the two experiments ranged between 0.0 to 96.7 and 0.0 to 100.0, respectively (Table 5). Accession, ME-0168 of Indonesian origin belonging to M. latifolia was found to be highly resistant (HR) against BRR (Fig. 6). The exotic accession ME-0168 was free from BRR symptoms and was on par with healthy control plants. Eight accessions that were found to be resistant (R) were G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489 and MI-0501. Whereas six accessions showed highly susceptible (HS) reaction, 12 were susceptible, and 18 were moderately resistant (MR) to BRR (Table 5, Fig. 7).

Discussion

Mulberry is affected by a variety of diseases produced by fungi and bacteria leading to severe yield loss and reducing the nutritive value of leaves. The repeated harvesting of leaves during the cultivation of mulberry leads to the depletion of soil nutrients and makes plants vulnerable to soil-borne diseases (Narayanan et al., 2015). Among the many fungal pathogens involved in causing root rot disease of mulberry, L. theobromae is a dominating fungus causing severe yield loss due to a decrease in plant population. L. theobromae has a broad range of plant hosts and is more prevalent in tropical and subtropical countries. Although several studies reported the pathogenic potential of this fungal species, it is also capable of surviving and spreading as an endophytic plant associate (de Silva et al., 2019; Muniz et al., 2011; Salvatore et al., 2020).
The study of micromorphological and cultural features is indispensable for the characterization of fungal isolates. In the case of genus Lasiodiplodia microscopic examination of conidia and paraphyses is important as these features differentiate various species of this genus (Rosado et al., 2016). Like several other research investigations, the present study also utilized SEM observations for interpreting micromorphological features of 20 L. theobromae isolates (Alves et al., 2008; Muniz et al., 2011; Tovar-Pedraza et al., 2012). All the sporulating isolates showed pycnidia with septate paraphyses, which is a characteristic feature of L. theobromae (Latha et al., 2013). The mature conidia appeared dark brown with thick wall and longitudinal striations. The mature conidial length was found in the range from 25 μm to 17 (± 3.0) μm and breadth was in the range from 14 μm to 7 (± 2.0) μm. PDA was frequently used to study the growth characteristics of L. theobromae isolates as this medium composition favored maximum growth of L. theobromae (Latha et al., 2013). L. theobromae isolates grew as irregular shaped colonies with greenish black/grey color and black color in reverse (Pečenka et al., 2021).
Based on cultural and morphological features, the isolated fungal pathogens were identified as L. theobromae. Twenty selected isolates representing four states of South India were subjected to prove Koch’s postulates which confirmed their pathogenicity with varying degrees of disease severity. However, the presence of highly varied cultural and morphological characteristics, diverse host range, ability to cause different symptoms and widespread geographical distribution of L. theobromae alarms about the existence of several strains in nature (Slippers et al., 2013; Sowmya et al., 2018; Xie et al., 2014). The molecular classification is rapid and provides precise phylogenetic distinctiveness.
Phylogenetic analysis of ITS and β-tubulin regions confirmed the identity of the selected isolates as L. theobromae. The 20 isolates of L. theobromae from this study, type strain (CBS 164.96) and reference sequences (CBS 111530 and COAD 1788) were grouped into a single cluster and separated from other known species of Lasiodiplodia. Even though the isolates were grouped into a single clade, four isolates (MRR-103, 143, 144, and 160) were away from the type strain (Fig. 4), the bootstrap values were low (53%) and the sequence homology of these four isolates was 98% as compared to the other isolates which had a high similarity of 100% with the type strain with high bootstrap values (83%). Likewise, Pappachan et al. (2020), isolated L. theobromae isolate from the infected roots of mulberry collected from Mizoram, Northeast India has 99.03% homologous to L. theobromae Bl16 (MK813947) with low bootstrap value.
Sowmya et al. (2018) based on cultural, morphological, pathogenicity, SSR and RAPD markers revealed significant variations among the L. theobromae isolates obtained from the infected gardens of Karnataka, Andhra Pradesh and Tamil Nadu. Also from her study, she could not establish a clear correlation between genetic diversity and geographical distribution. Many researchers in other crops, identified different species of Lasiodiplodia, however, from our studies, we could only identify L. theobromae causing BRR of mulberry in India (Pappachan et al., 2020; Radhakrishnan et al., 1995; Sowmya et al., 2018; Sukumar and Padma 1999) and this is the first detailed phylogenetic analysis of L. theobromae causing BRR of mulberry in India using two genes.
The wild species of mulberry (M. serrata and M. laevigata) are known to possess several important resistant genes to many biotic and abiotic factors (Tikader and Dandin, 2007; Vijayan et al., 2011). Even though 68 species from Morus were available, only a limited number of species have been utilized in developing mulberry varieties (Datta, 2000). The emerging new varieties from the narrow gene pool are more homogenous and become HS to pathogens and vulnerable to poor environmental conditions (Vijayan et al., 2011). Cultivation of disease resistant varieties is one of the best approaches to exclude plant pathogens. The selection of promising genotypes will contribute to developing resistant breeding methods.
Screening of mulberry germplasm using pathogenic L. theobromae isolates, and identification of highly durable BRR resistant varieties can aid in achieving sustainable sericulture. Mulberry researchers in India have identified resistant accessions to bacterial leaf spot (Xanthomonas campestris), powdery mildew (Phyllactinia corylea), charcoal root rot (M. phaseolina), and root knot (Arunakumar et al., 2021; Banerjee et al., 2009; Chattopadhyay et al., 2010; Maji, 2011; Pinto et al., 2018). To our knowledge, there is no report on screening of germplasm resistance against L. theobromae causing BRR. To select promising mulberry germplasm against BRR, the present study attempted to evaluate 45 diverse accessions following the root dip method of inoculation under glasshouse conditions. In the repeated experiments, the mulberry accession ME-0168 which is an Indonesian origin was consistently found to be HR to BRR. Other eight accessions, G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489, and MI-0501 were found to be resistant.
Most of the resistant accessions, ME-0006, ME-0011, ME-0093, including HR (ME-0168) and MR (ME-0066 and ME-0141) were from exotic collections and belonged to M. latifolia. This indicates M. latifolia possesses resistant genes against BRR of mulberry. The other four resistant accessions, MI-0006, MI-0291, MI-0489, and MI-0501, belonged to M. indica. Joty et al. (2019) found that the leaf extracts of M. indica and M. latifolia induced the maximum antibacterial and antioxidant activity against the pathogenic bacteria. Interestingly, G2 high yielding mulberry variety was found to be resistant to BRR is a hybrid of ME-0168 and S-34, similarly, G4 which is MR to BRR is a cross pollinated hybrid of ME-0168 and S-30. This proves that the identified HR accession ME-0168 along with the other promising resistant sources can be exploited in mulberry breeding for developing BRR resistant varieties and to develop mapping populations which successively helps in the identification of molecular markers associated with BRR.
In conclusion, the outcomes of this research represent the detailed study of phylogeny and pathogenicity of L. theobromae causing BRR of mulberry in India. The MRR-142 isolate, isolated from Ananthapur, Andhra Pradesh was found to be highly pathogenic which caused a maximum percentage of wilting and rotting. Further, the same isolate was used to screen resistant accessions. The root dip inoculation method identified the HR accession ME-0168 against BRR of mulberry. ME-0168, along with the other sources of resistance can be utilized as resistant parents for developing BRR resistance varieties.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

The first author would like to thank the Department of Science and Technology, for providing Ramanujan Fellowship. This study was funded by Science and Engineering Research Board (SERB), New Delhi, Grant (SERB SB/S2/RJN-049/2015). The authors also thank Scientists of different Regional Sericultural Research Stations and Research Extension Centers of the Institute for their support during the survey.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

Fig. 1
Black root rot disease symptoms on mulberry plant under field conditions. (A) Early symptoms on a young mulberry plant, yellowing and wilting of leaves. (B) Severely infected root and appearance of newly regenerated roots. (C) Blackening and discoloration of the vascular tissue of the infected plant.
ppj-oa-01-2022-0005f1.jpg
Fig. 2
Colony characteristics of Lasiodiplodia theobromae isolated from roots of mulberry. (A) Seven-day-old white to light-cream colony (upper surface). (B) Lower surface. (C) Fourteen-day-old aged dark grey colonies with fluffy aerial mycelium. (D) Production of liquid exudates.
ppj-oa-01-2022-0005f2.jpg
Fig. 3
Conidial morphological characteristics observed under light microscope and scanning electron microscopy (SEM) for Lasiodiplodia theobromae isolate MRR-142. (A, B) Mycelia and conidia. (C, D) Hyaline, immature conidia. (E, F) Mature conidia with septa. (G-I) Observation under SEM. Scale bars = 50 μm (A-C, E-G), 20 μm (D, H, I).
ppj-oa-01-2022-0005f3.jpg
Fig. 4
Phylogenetic tree generated from maximum likelihood method and Tamura 3-parameter model (T92) model based on combined internal transcribed spacer region and Beta tubulin genes. Bootstrap values (≥50%) from 1,000 replicates are indicated at the nodes. The type strains are indicated with boldface type (T) and the tree was rooted with Barriopsis fusca.
ppj-oa-01-2022-0005f4.jpg
Fig. 5
Pathogenicity test results of Lasiodiplodia theobromae isolate, MRR-142 on susceptible mulberry accession Victory-1 and Thailand male.
ppj-oa-01-2022-0005f5.jpg
Fig. 6
Highly resistant (ME-0168) (A) and highly susceptible (MI-0037) (B) mulberry accessions, evaluated using pathogenic isolate, MRR-142 of Lasiodiplodia theobromae.
ppj-oa-01-2022-0005f6.jpg
Fig. 7
Disease reaction of mulberry accessions against black root rot. HR, highly resistant; R, resistant; MR, moderately resistant; S, susceptible; HS, highly susceptible, P < 0.001.
ppj-oa-01-2022-0005f7.jpg
Table 1
Disease rating scale for black root rot reaction
Disease severity index Root rot (%) Root wilt (%) Pathogenicity Disease reaction
1 No rotting No wilting Nil Highly resistant
2 1-25 rotting 1-25 wilting Low Resistant
3 26-50 rotting 26-50 wilting Moderate Moderately resistant
4 51-75 rotting 51-75 wilting High Susceptible
5 >75 rotting >75 wilting Very high Highly susceptible
Table 2
List of mulberry accessions used for evaluating black root rot under glasshouse conditions
Accession no. Germplasm Species Country Genetic origin
ME-0033 Thailand Male M. alba Thailand Selection
ME-0106 China Black-B M. alba China Collection
ME-0239 BR-8 M. alba Thailand Collection
MI-0017 Sujanpur-5 M. alba India, Punjab OPH selection
MI-0026 Punjab Local M. alba India, Punjab OPH selection
MI-0254 UP-22 M. alba India, Uttar Pradesh Clonal selection
MI-0643 L-2 M. alba India, Uttar Pradesh Collection
MI-0828 Hosur-C8 M. alba India, Tamil Nadu CPH
ME-0254 M. cathayana (hybrid) M. cathayana - Developed
MI-0006 LF-1 M. indica India, Uttar Pradesh Clonal selection
MI-0007 Himachal Local M. indica India, Himachal Pradesh Selection
MI-0037 S-799 M. indica India, West Bengal OPH selection
MI-0043 S-523 M. indica India, West Bengal OPH selection
MI-0052 Mysore local M. indica India, Karnataka OPH selection
MI-0068 Kajali M. indica India, West Bengal Clonal selection
MI-0120 S-642 M. indica India, Karnataka OPH selection
MI-0124 S-763 M. indica India, West Bengal OPH selection
MI-0172 S-1708 M. indica India, West Bengal OPH selection
MI-0267 ERRC-215 M. indica India, Kerala Collection
MI-0271 ERRC-123 M. indica India, Kerala Collection
MI-0275 ERRC-103 M. indica India, Kerala Collection
MI-0277 ERRC-106 M. indica India, Kerala Collection
MI-0285 C-6 M. indica India, Tamil Nadu Collection
MI-0291 C-18 M. indica India, Tamil Nadu Collection
MI-0308 Victory-1 M. indica India, Karnataka CPH
MI-0347 Moulai M. indica India, Meghalaya Collection
MI-0421 Pillighat M. indica India, Madhya Pradesh Collection
MI-0489 Vadapuram M. indica India, Kerala Survey
MI-0501 Meghamalai-1 M. indica India, Tamil Nadu Survey
MI-0520 YERCAUD-3 M. indica India, Tamil Nadu Survey
MI-0523 Cuckpilla M. indica India, Karnataka Collection
MI-0551 Kollihills-1 M. indica India, Tamil Nadu Survey
MI-0580 School Salem M. indica India, Tamil Nadu Survey
MI-0682 Kota-4 M. indica India, Rajasthan Survey
ME-0006 M. multicaulis M. latifolia Indonesia Collection
ME-0011 Philippines M. latifolia Philippines Clonal selection
ME-0066 Kousen M. latifolia Japan Selection
ME-0093 Australia M. latifolia Australia Collection
ME-0141 Roso M. latifolia Japan Collection
ME-0168 M. multicaulis M. latifolia Indonesia Collection
ME-0008 M. nigra M. nigra Indonesia Selection
ME-0176 Reblaira M. rotundiloba France Collection
G2 G2 Morus sp. India, Karnataka CPH
G4 G4 Morus sp. India, Karnataka CPH
RC-2 RC-2 Morus sp. India, Karnataka Selection

OPH, open pollinated hybrids; CPH, cross pollinated hybrids.

Table 3
GenBank accession numbers of ITS and β-tubulin of Lasiodiplodia spp. used in the phylogenetic analyses
Species Isolate Host Country ITS β-tubulin
L. citricola IRAN 1522C Citrus sp. Iran GU945354 KU887505
L. citricola IRAN 1521C Citrus sp. Iran GU945353 KU887504
L. crassispora WAC12533 Santalum album Australia DQ103550 KU887506
L. crassispora CMW 13488 Eucalyptus europhylla Venezuela DQ103552 KU887507
L. euphorbicola CMM 3609 Jatropha curcas Brazil KF234543 KF254926
L. euphorbicola CMM 3652 Jatropha curcas Brazil KF234554 KF254938
L. mahajangana CMW 27801 Terminalia catappa Madagascar FJ900595 FJ900630
L. mahajangana CMW 27818 Terminalia catappa Madagascar FJ900596 FJ900631
L. missouriana UCD 2193MO Vitis vinifera USA HQ288225 HQ288304
L. missouriana UCD 2199MO Vitis vinifera USA HQ288226 HQ288305
L. pseudotheobromae CBS 116459 Gmelina arborea Costa Rica EF622077 EU673111
L. pseudotheobromae CMM 3887 Jatropha curcas Brazil KF234559 KF254943
L. subglobosa CMM 3872 Jatropha curcas Brazil KF234558 KF254942
L. subglobosa CMM 4046 Jatropha curcas Brazil KF234560 KF254944
L. theobromae CBS 164.96 Fruit New Guinea AY640255 KU887532
L. theobromae CBS 111530 Unknown Unknown EF622074 KU887531
L. theobromae COAD 1788 Cocos nucifera Brazil KP244698 KP308528
L. theobromae MRR-002 Morus sp. India, AP KY884641 MW287586
L. theobromae MRR-017 Morus sp. India, KA KY964307 MW287587
L. theobromae MRR-030 Morus sp. India, KA MW282879 MW287588
L. theobromae MRR-056 Morus sp. India, KA MW282880 MW287589
L. theobromae MRR-073 Morus sp. India, KA MW282881 MW287590
L. theobromae MRR-091 Morus sp. India, KA MW282882 MW287591
L. theobromae MRR-100 Morus sp. India, KA MT075437 MW114665
L. theobromae MRR-102 Morus sp. India, KA MT075438 MW114666
L. theobromae MRR-103 Morus sp. India, KA MT075439 MW114667
L. theobromae MRR-126 Morus sp. India, TN MT075440 MW114668
L. theobromae MRR-127 Morus sp. India, TN MT075441 MW114669
L. theobromae MRR-128 Morus sp. India, TN MT075442 MW114670
L. theobromae MRR-130 Morus sp. India, TN MT075443 MW114671
L. theobromae MRR-133 Morus sp. India, TN MW282883 MW287592
L. theobromae MRR-142 Morus sp. India, AP MT075444 MW114672
L. theobromae MRR-143 Morus sp. India, AP MT075445 MW114673
L. theobromae MRR-144 Morus sp. India, AP MT075446 MW114674
L. theobromae MRR-153 Morus sp. India, TS MT075447 MW114675
L. theobromae MRR-160 Morus sp. India, TS MT075448 MW114676
L. theobromae MRR-161 Morus sp. India, TS MW282884 MW287593
L. viticola UCD 2553AR Vitis vinifera USA HQ288227 HQ288306
L. viticola UCD 2604MO Vitis vinifera USA HQ288228 HQ288307
Barriopsis fusca CBS 174.26 Citrus sp. Cuba KF766149 EU673109

Extype isolates are in bold.

ITS, internal transcribed spacer; MRR, mulberry root rot samples collected from different states of India; AP, Andhra Pradesh; KA, Karnataka; TN, Tamil Nadu; TS, Telangana.

Table 4
Means comparison of black root rot disease scores of Lasiodiplodia theobromae isolates on two susceptible mulberry genotypes
Place Isolates Victory-1 Thailand Male Pathogenicity


Wilting (%) Rotting (%) Wilting (%) Rotting (%)
India, AP MRR-002 77.0 ± 2.0 aba 72.3 ± 6.6 abc 75.3 ± 11.2 abc 80.3 ± 14.1 abc Very high
India, KA MRR-017 93.3 ± 7.6 a 90.0 ± 5.0 ab 88.6 ± 10.0 ab 88.0 ± 10.5 abc Very high
India, KA MRR-030 52.0 ± 6.0 c 57.0 ± 11.0 c 66.6 ± 11.5 bc 60.3 ± 4.7 c Very high
India, KA MRR-056 13.0 ± 3.6 d 11.3 ± 3.5 d 13.6 ± 8.3 e 13.6 ± 7.7 d Low
India, KA MRR-073 77.6 ± 11.0 ab 81.3 ± 8.3 abc 87.0 ± 8.5 ab 89.6 ± 7.5 abc Very high
India, KA MRR-091 87.0 ± 2.6 ab 80.3 ± 5.5 abc 93.0 ± 5.0 ab 93.6 ± 7.7 ab Very high
India, KA MRR-100 64.0 ± 11.1 bc 57.0 ± 10.1 c 62.3 ± 8.5 bc 64.0 ± 9.6 bc Very high
India, KA MRR-102 69.0 ± 9.0 bc 64.3 ± 19.8 bc 47.3 ± 30.0 cd 66.6 ± 32.1 abc Very high
India, KA MRR-103 85.6 ± 10.0 ab 88.0 ± 10.0 abc 92.6 ± 11.0 ab 93.3 ± 7.6 ab Very high
India, TN MRR-126 14.6 ± 3.0 d 9.3 ± 4.1 d 17.3 ± 2.5 de 20.0 ± 2.0 d Low
India, TN MRR-127 82.6 ± 13.6 ab 73.0 ± 15.8 abc 91.0 ± 3.6 ab 92.0 ± 3.4 ab Very high
India, TN MRR-128 84.0 ± 6.5 ab 88.6 ± 9.0 abc 88.3 ± 9.4 ab 90.3 ± 4.0 ab Very high
India, TN MRR-130 68.6 ± 14.4 bc 61.6 ± 17.0 bc 71.0 ± 3.0 abc 72.0 ± 6.5 abc Very high
India, TN MRR-133 8.0 ± 4.0 d 4.6 ± 1.5 d 16.6 ± 3.0 de 17.0 ± 1.0 d Low
India, AP MRR-142 97.6 ± 2.5 a 100.0 ± 0.0 a 99.3 ± 1.1 a 95.0 ± 5.0 a Very high
India, AP MRR-143 78.6 ± 3.5 ab 79.0 ± 20.0 abc 88.0 ± 3.0 ab 86.6 ± 5.7 abc Very high
India, AP MRR-144 79.0 ± 4.5 ab 83.3 ± 7.0 abc 77.3 ± 11.5 abc 80.6 ± 9.5 abc Very high
India, TS MRR-153 3.6 ± 1.5 d 5.6 ± 2.5 d 7.6 ± 1.5 e 10.3 ± 0.5 d Low
India, TS MRR-160 81.6 ± 9.4 ab 66.6 ± 7.3 bc 72.0 ± 16.8 abc 87.6 ± 4.1 abc Very high
India, TS MRR-161 85.0 ± 6.0 ab 78.3 ± 14.0 abc 90.0 ± 5.5 ab 87.6 ± 2.5 abc Very high
Control 0.0 ± 0.0 d 0.0 ± 0.0 d 0.0 ± 0.0 e 0.0 ± 0.0 d

a The values with the same letter do not differ significantly as per the Tukey test (P < 005).

Table 5
DR of mulberry accessions against Lasiodiplodia theobromae using root dip method of inoculation under glasshouse conditions
Accession no. Experiment 1 Experiment 2


Wilting Rotting DR DSI ± SD Wilting Rotting DR DSI ± SD
ME-0033 79.13 75 HS 4.5 ± 0.6 77.52 87.58 HS 4.5 ± 0.6
ME-0106 44.34 38.8 MR 2.5 ± 0.6 31.07 27.12 MR 2.8 ± 0.5
ME-0239 34.79 20.2 MR 2.8 ± 0.5 41.53 31.51 MR 3.3 ± 0.5
MI-0017 38.98 29.7 MR 3.0 ± 0.0 36.79 31.65 MR 3.0 ± 0.0
MI-0026 27.74 39.2 MR 2.3 ± 0.5 41.57 36.64 MR 3.0 ± 0.0
MI-0254 27.83 25.5 MR 2.8 ± 0.5 28.92 37.83 MR 2.8 ± 0.5
MI-0643 60.84 68.8 S 3.8 ± 1.0 67.6 71.5 S 3.8 ± 0.5
MI-0828 28.26 37.9 MR 2.3 ± 0.5 42.93 20.09 MR 3.3 ± 0.5
ME-0254 26.72 27.6 MR 2.5 ± 0.6 70.71 71.15 S 4.3 ± 0.5
MI-0006 22.92 19.4 R 2.3 ± 0.5 21.49 16.49 R 2.3 ± 0.5
MI-0007 3.85 23.4 R 1.0 ± 0.0 28.75 41.61 MR 2.5 ± 0.6
MI-0037 75.55 88.5 HS 4.8 ± 0.5 78.57 88.39 HS 4.8 ± 0.5
MI-0043 58.75 63.7 S 3.5 ± 0.6 60.9 74.23 S 4.5 ± 0.6
MI-0052 53.2 64.8 S 3.8 ± 0.5 61.79 53.96 S 4.0 ± 1.2
MI-0068 30.63 38.8 MR 2.8 ± 0.5 49.79 39.87 MR 3.3 ± 0.5
MI-0120 51.34 66.8 S 3.5 ± 0.6 65.35 58.61 S 4.3 ± 0.5
MI-0124 71.19 74.6 S 4.3 ± 1.0 70.84 71.67 S 4.3 ± 0.5
MI-0172 67.56 71.6 S 4.3 ± 0.5 74.92 59.17 S 4.5 ± 0.6
MI-0267 62.47 62.9 S 3.8 ± 1.0 47.68 65.07 S 3.5 ± 0.6
MI-0271 71.13 57.5 S 4.3 ± 1.0 67.71 72.76 S 3.8 ± 0.5
MI-0275 32.51 32 MR 2.8 ± 0.5 30.83 39.21 MR 2.5 ± 0.6
MI-0277 30.63 29.9 MR 2.3 ± 0.5 55.32 52 S 4.0 ± 0.0
MI-0285 43.81 35.2 MR 3.3 ± 0.5 43.41 34.41 MR 3.0 ± 0.0
MI-0291 23.44 11.9 R 2.5 ± 0.6 17.26 15.17 R 1.8 ± 0.5
MI-0308 78.8 77.6 HS 4.8 ± 0.5 83.85 89.28 HS 5.0 ± 0.0
MI-0347 96.67 100 HS 5.0 ± 0.0 91.88 93.75 HS 5.0 ± 0.0
MI-0421 60.55 37.2 S 3.8 ± 0.5 52.91 48.04 S 3.8 ± 0.5
MI-0489 16.95 25 R 2.0 ± 0.0 23.33 21.36 R 2.3 ± 1.0
MI-0501 19.71 23.6 R 2.5 ± 0.6 24.8 16.88 R 2.5 ± 0.6
MI-0520 32.23 29.2 MR 2.5 ± 0.6 27.72 31.28 MR 2.5 ± 0.6
MI-0523 35.99 27.1 MR 3.0 ± 0.0 36.93 34.56 MR 3.0 ± 0.0
MI-0551 32.41 32.8 MR 2.5 ± 0.6 32.44 34.08 MR 2.5 ± 1.0
MI-0580 44.6 37.9 MR 3.3 ± 0.5 58.51 69.5 S 3.8 ± 0.5
MI-0682 31.75 26.5 MR 2.8 ± 0.5 43.02 30.21 MR 3.0 ± 0.0
ME-0006 0 11.8 R 1.0 ± 0.0 6.95 9.57 R 1.8 ± 0.5
ME-0011 15.82 18 R 1.8 ± 0.5 24.14 19.28 R 2.5 ± 0.6
ME-0066 31.05 33.1 MR 2.8 ± 0.5 39.28 35.38 MR 3.3 ± 0.5
ME-0093 15.03 8.9 R 2.0 ± 0.0 18.17 13.37 R 2.0 ± 0.0
ME-0141 26.52 28.1 MR 2.5 ± 0.6 31.25 28.5 MR 2.8 ± 0.5
ME-0168 0 0 HR 1.0 ± 0.0 0 0 HR 1.0 ± 0.0
ME-0008 51.17 54.3 S 3.3 ± 0.5 79.15 75.6 HS 4.8 ± 0.5
ME-0176 27.26 30 MR 2.8 ± 0.5 19.92 11.64 R 2.3 ± 0.5
G2 4.52 16.3 R 1.5 ± 0.6 13.54 10.68 R 1.8 ± 0.5
G4 26.43 33.8 MR 2.3 ± 0.5 27.71 27.98 MR 2.8 ± 0.5
RC-2 76.89 75.4 HS 4.5 ± 0.6 80.17 92.14 HS 4.8 ± 0.5

Disease reaction (DR) of the germplasm accessions were categorized according to the modified scale (Ahmed et al., 1999). DF = 44, P < 0.001. DSI, disease severity index; SD, standard deviation; HR, highly resistant; R, resistant; MR, moderately resistant; S, susceptible; HS, highly susceptible.

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