Background

Dalbergia sissoo Roxb. ex DC., commonly known as shisham, is a valuable timber species in Fabaceae family, native to Afghanistan, Bangladesh, Bhutan, India, Iraq, Iran, Myanmar, Nepal, and Pakistan and introduced into Africa, Australia, China, and the USA [1,2,3]. It is a pioneer tree of primary succession in natural riverine forest of rivers Indus, the Ganges, Yamuna and Brahmaputra with their tributaries and also an important multipurpose tree growing outside forest which yields beautiful dark brown wood for furniture and panels, additionally used for strong poles, quality fodder, fuel wood and folk medicine [4]. The species is a suitable tree for wheat-based agroforestry system as it is deciduous and fixes nitrogen [5, 6].

Widespread mortality in D. sissoo (Fig. 1) has been reported from many parts of India [7,8,9,10,11,12,13,14] as well as from Bangladesh [15], Nepal [16], and Pakistan [17], and one study in India estimated a loss equivalent to US$ 200 million due to mortality of 400,000 mature trees [12, 18]. Fusarium solani (Mart.) Sacc. f. sp. dalbergiae Bakshi and Singh (hereafter referred as Fsd) has been reported as one of the primary pathogenic fungi causing mortality in D. sissoo due to vascular wilt which is prevalent in the riverine plains of India, Pakistan, Nepal, and Bangladesh [8]. Other fungal pathogens viz., Fusarium oxysporum, Oxyporus latemarginatus [19], Ganoderma lucidum (Curtis) P. Karst [20], Cercospora sissoo [21], Colletogloeum sissoo, Glomerella cingulata and Septothyrella dalbergiae [22], Mycosphaerella dalbergiae [23], Phyllachora dalbergiae (=Phyllachora viventis; [24]), Phyllactinia dalbergiae [25], Maravalia achroa and Uredo sissoo [26], Ceratocystis manginecans M. Van Wyk, Al Adawi & M. J. Wingf [27], and Lasiodiplodia theobromae (Pat.) Griffon and Maubl [28]. have also been reported for causing diseases in D. sissoo.

Fig. 1
figure 1

D. sissoo completely wilted (a) and healthy (b) trees in their natural habitat (riverine forest) Thano forest range, Dehra Dun, India

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Tree improvement programs in developing world have largely been focused on productivity improvement of timber and to a lesser extent on disease resistance [29]. Timber tree species like D. sissoo have a rotation age of 30 years or more which has also added to the problem of screening for disease resistance in the field despite widely reported infections and hence, necessitates finding a quicker and reliable system to identify disease-resistant genotypes of D. sissoo. Screening and selection of disease-resistant clones by in vitro co-culturing under gnotobiotic conditions of desired cloned genotypes with the fungal pathogen or toxin is an important crop improvement tool and has been adopted by different researchers [30,31,32,33,34,35,36]. In vitro growth condition is not only highly reproducible but also minimize the influence of external abiotic and biotic factors and ensures homogenous interaction between in vitro cultures of desirable host genotypes and inoculums of only known microbe giving a perfect gnotobiotic condition and thus, evincing a robust system to study plant-microbe interactions and facilitates estimation of resistance of desirable host plant genotypes [37, 38]. In the present study, callus and plantlets of ten genotypes of D. sissoo were co-cultured in vitro along with virulent strains of Fsd in a dual culture setup. This in vitro screening of D. sissoo genotypes against virulent strains of Fsd was aimed at studying the variability and selection of resistant D. sissoo genotypes which, therefore, can be used as an indirect selection method for disease-resistant genotypes of tree species [39].

Methods

Plant material

Phenotypically superior trees of D. sissoo have been selected from various parts of northern India, cloned through branch cuttings are being maintained in clonal multiplication block. Ten genotypes that were part of long-term field trials for improved productivity were selected for this study (Table 1). Genotype 14, a commercial cultivar of D. sissoo resistant to shisham die-back was also chosen for the study [40].

Table 1 Source location of selected genotypes of D. sissoo in the study
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Fungal material

F. solani f. sp. dalbergiae strains (isolate no. 1145 and 1149) were procured from the National Type Culture Collection (NTCC) and were cultured in Petri dishes (9 cm) on potato dextrose agar (PDA) medium at 25 ± 2 °C.

In vitro establishment of aseptic cultures of D. sissoo

Explant sterilization

Healthy and uninfected nodal segments of the selected genotypes of D. sissoo were used for aseptic culture establishment. Explants were washed in running tap water followed by soaking in a 0.5 % solution of antiseptic Cetrilak® (Cetrimide 5% w/v, India) and then in an aqueous solution of 0.1 % Bavistin® (Carbendazim WP; 50 % w/v, India). Surface sterilization of explants was carried out in a laminar airflow with 0.1% Mercuric chloride [41, 42].

Axillary bud proliferation and in vitro rooting

Nodal segments of the branch containing single axillary bud from each accession were collected, sterilized, and inoculated. For bud induction and multiplication, best-responded treatments, Murashige and Skoog (MS) medium [43] comprising 4.44 μM 6-Benzylaminopurine (BAP) + 2.69 μM 1-Naphthaleneacetic acid (NAA) and MS medium comprising 4.44 μM BAP + 1.34 μM NAA, were used, respectively [41]. Cultures were maintained on standardized multiplication medium at 5 weeks interval and repeated further in subsequent sub-culturing. Micro shoots of size > 2.5 cm were excised and transferred for root induction in half strength MS medium supplemented with Indole-3-butyric acid (IBA) at 4.92 μM concentrations [41]. Culture medium pH was adjusted to 5.8 and autoclaved for 15 min at 121 °C and 1.0 × 105 Pa. Incubation temperature of culture room was 25 ± 2 °C and 55 ± 5 % relative humidity under a 16/8 hr (light/dark) photoperiod with light supplied by cool-white fluorescent tubes (Philips, India) at an intensity of 35 μmoles/m2/s.

Callus induction

Nodal explants of D. sissoo were collected from each genotype and sterilized as mentioned earlier, further inoculated on MS medium supplemented with BAP (2.22 - 6.66 μM) alone or in combination with 2,4-Dichlorophenoxyacetic acid (2,4-D; 2.26 - 6.79 μM) for callus induction. The callus was maintained on MS medium supplemented with 4.44 μM BAP and 2.69 μM NAA in culture condition as mentioned above for further in vitro screening [41].

In vitro screening and selection of resistant D. sissoo genotypes against Fsd

Fungal inoculum preparation

A mycelial disc (4 mm dia) from growing margins of the Fsd culture was transferred to an Erlenmeyer flask (250 ml) containing 100 ml Carboxy Methyl Cellulose (CMC) medium [44] for sporulation. The culture was incubated for 15 days at 25 ± 2 °C and then viewed in a hemocytometer slide for conidial count. Consequential conidial suspension was diluted to the desired concentration (1 × 101, 1 × 103, and 1 × 105 conidia/ml) in the appropriate inoculation medium.

Screening of callus against Fsd

Callus of each genotype of D. sissoo was inoculated on a standardized multiplication medium. On growing callus 5 μl droplet of conidial suspensions of Fsd (1145 and 1149) at three concentrations (1 × 101, 1 × 103 and 1 × 105 conidia/ml) were inoculated atop the center of the callus and incubated at 25 °C ± 2 as described before. After inoculation of callus tissue with conidial suspensions of Fsd, the extent of infection was assessed on the 9th day by measuring the diameter of fungal growth on the tissue as well as conditions of callus, i.e., either dead or alive. Fungal growth (cm) was measured by taking the average of diameters at both X-X’ and Y-Y’axes of spread. Means of fungal radial diameter were analyzed using a parametric test.

Screening of plantlets against Fsd

In vitro rooted plantlets of each genotype of D. sissoo, were inoculated with Fsd (1145 and 1149) conidia at 1 × 105 conidia/ml concentration in 0.1% water agar supplemented with MS salts, and incubated in culture conditions as described before. The plantlets were infected only with the highest Fsd conidial concentration as the differences during in vitro screening of callus of ten D. sissoo genotypes were notably evident at this concentration. The extent of infection was assessed at regular intervals throughout a 15-day period of incubation using the following disease scores: 0 = healthy plant, l = main root tip necrotic, 2 = whole root system infected, 3 = stem infected and appearance of wilt symptoms, 4 = whole plant wilted, and 5 = plant dead. Infection extent of each genotype was scored from 0 to 5 at the end of 5th, 7th, 9th, 11th, 13th, and 15th days. On each observed day, means of infection extent score of genotypes were compared using non-parametric test.

Experimental design and statistical analyses

The experiments were laid in completely randomized design (CRD) with five replicates for each treatment and normal data were analyzed using analysis of variance (ANOVA) in Genstats 5 edition 3.2 for PC/Windows NT (Copyright 1995, LAWES Agricultural Trust (Rothamsted Experimental Station) and means were compared with least significant difference (LSD). Non-normal data analyzed using Kruskal-Wallis (KW) test in SPSS statistics-23. Results with a significant difference were KW test compared using rank corresponding to mean of infection extent of plantlet of genotype on each observed day. The genotype having rank with low numerical value were resistant whereas genotype having rank with high numerical were susceptible against the inoculated isolate of Fsd.

Results

In vitro response of callus of ten genotypes of D. sissoo against infection to Fsd

Results of the experiment suggest that Fsd isolates (1145 and 1149), D. sissoo genotypes, and the interaction between them had significantly affected the extent of fungal infection on the callus. High concentration (1 × 105 conidia/ml) of both isolates resulted in maximum spread of fungus 3.12 cm and 3.14 cm, respectively, on callus after 9 days whereas low concentration (1 × 101 conidia/ml) of both isolates resulted in minimum spread of fungus on callus 1.04 cm and 1.28 cm, respectively. Also, the extent of fungal infection on callus caused by each treatment differed significantly. Effect of D. sissoo genotypes on the growth of Fsd mycelium on callus was significant; fungal spread of both Fsd isolates 1145 and 1149 was least on callus of genotype 14 (0.9 cm and 1.16 cm, respectively) whereas genotypes 232 and 41 could not restrain the fungal spread of isolates 1145 and 1149, respectively, and thus, resulted in maximum spread of 2.90 cm and 2.89 cm, respectively. Fungal growth on callus of genotype 66 for Fsd isolates 1145 and 1149 was 1.00 cm and 1.23 cm, respectively, which is at par with genotype 14 (Table 2, Fig. 2a–d). Among the genotypes, callus of genotypes 14 and 66 showed resistance to fungal growth of both the Fsd isolates.

Table 2 Effect of conidial concentration of Fsd (1145 and 1149) on callus of D. sissoo genotypes
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Fig. 2
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a Effect of Fsd isolate 1145 on in vitro raised callus of D. sissoo genotypes, from left to right genotype 19, 237, 204, 14, 66, 41, 232, 10, 24, and 20. b Callus of genotype 14 inoculated with Fsd (1149) after 30 days of infection. c Callus of genotype 66 inoculated with Fsd (1145) after 30 days of infection. d Control for (a). e Control for (f and g; from left to right genotypes 14, 66, 232, 237, 204, 24, 19, 41, 201 and 10). f Effect of Fsd isolate 1145 on in vitro raised plantlets of ten genotypes of D. sissoo (from left to right genotypes 14, 66, 232, 237, 204, 24, 19, 41, 201 and 10). g Effect of Fsd isolate 1149 on in vitro raised plantlets of ten genotypes of D. sissoo (from left to right genotypes 14, 66, 232, 237, 204, 24, 19, 41, 201 and 10). h Plantlets of genotype 14 infected with both isolates of Fsd 1149 (left most test tube) and 1145 (second test tube from left) and uninfected control (third test tube from left) after 3 weeks of infection. i Plantlets of genotype 66 infected with both isolates of Fsd 1149 (left most test tube) and 1145 (second test tube from left) and uninfected control (third test tube from left) after 3 weeks of infection

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Observations on the interaction between host genotypes and conidial concentrations of Fsd isolates (1145 and 1149) revealed that maximum spread of fungus was 5.02 cm and 4.68 cm, respectively, on callus of genotype 232 at 1 × 105 conidia/ml concentration whereas a minimum spread of fungal growth 0.77 cm and 1.06 cm, respectively, was observed on callus of genotype 14 inoculated with 1 × 101 conidia/ml concentration. An increase in Fsd mycelium spread was observed in callus of genotype 232 from 1.42 to 5.02 cm upon increasing conidial concentration of isolate 1145 from 1 × 101 to 1× 105 conidia/ml and similarly from 1.53 to 4.68 cm for isolate 1149. On the other hand, genotype 14 apparently restricted fungal growth of isolate 1145 on callus as no significant difference in fungal spread was observed when conidial concentration was increased from 1 × 101 to 1 × 105 conidia/ml. However, for isolate 1149 an increase in conidial concentration from 1 × 101 conidia/ml to 1 × 105 conidia/ml significantly affected the fungal spread on callus of genotype 14. It was interesting to note that the fungal spread on callus of genotype 14 was non-significant when concentration was increased from 1 × 101 to 1 × 103 conidia/ml as well as from 1× 103 conidia/ml to 1× 105 conidia/ml. Genotype 66, though not as promising as genotype 14, showed some resistance as fungal spread on callus was non-significant when conidial concentration of both isolates was increased from 1 × 101 to 1 × 103 conidia/ml, however fungal spread on callus differed significantly when conidial concentration of both isolates was increased from 1 × 103 conidia/ml to 1 × 105 conidia/ml. In other genotypes spread on callus differed significantly when conidial concentration of both isolates was increased from 1 × 101 to 1 × 103 conidia/ml as well as from 1 × 103 to 1× 105 conidia/ml (Table 2, Fig. 2a–d).

In vitro response of plantlets of ten genotypes of D. sissoo against infection of Fsd

Plantlets of ten genotypes of D. sissoo were screened in vitro against two isolates of Fsd and observation was assessed using a disease score. Disease score data was non-normal so KW test was applied and significantly varying treatments were compared using rank. Results suggest that genotype 14 ranked first on all observed days except on day seven, where it was a joint second against isolate 1149. The corresponding mean disease score of genotype 14 on day five was 0.4 against both isolates which increased to 2.4 and 2.6 against isolates 1145 and 1149, respectively, on the 15th day. Genotype 66 ranked second consistently on all observed days against both isolates and its corresponding mean disease score was 0.6 on the 5th day which grew to 2.8 on the 15th day against both isolates. Genotype 19 managed rank 3.5 and 3.0 against 1145 and 1149, respectively, on the 5th day, 3.0 against both isolates on the 9th day, 11th day, and 13th day but rank 6.5 on the 15th day. The corresponding mean disease score was 0.8 on day 5th which steeply reached to 5.0, which meant completely dead plantlets, on the 15th day, which suggests that slight resistance was shown by genotype during initial days of treatment but was lost by the 15th day. Similar results were obtained for genotype 41.

Plantlets of susceptible genotypes were prone to infection from the beginning and maintained susceptibility throughout the observation. Genotypes 10, 24, 201, 204, 232, and 237 on the 5th day had mean disease score more than one and their relative ranks were 6, 8.5, 6.0, 10, 6, and 8.5, respectively, against isolates 1145 whereas against isolate 1149 their ranks were 5, 8, 8, 8, 8 and 8, respectively. Five genotypes 24, 201, 204, 232, and 237 scored more than three against isolate 1149 on 7th day and their corresponding ranks were 8, 8, 8, 8, and 10, respectively, though against isolate 1145 genotypes 10, 24, 201, 204, 232, and 237 had mean disease scores of 1.8, 2.0, 1.0, 1.8, 1.0, and 2.4, respectively, and the corresponding ranks were 7.5, 9, 4, 7.5, 4, and 10, respectively. On the 11th day, genotypes 24, 232, and 237 had the highest mean disease scores of 5 and the corresponding ranks were 9 against isolate 1149 whereas the same genotypes had mean disease scores of 4, 4, and 4.2, respectively, with the corresponding ranks 8.5, 8.5, and 10, respectively, against isolates 1145. Genotypes 10, 24, 201, 204, 232, and 237 had a mean disease score of 5 on the 13th and 15th day against both isolates (Table 3, Fig. 2e–i).

Table 3 Disease scoring of plantlets of ten D. sissoo genotypes inoculated with Fsd (1145 and 1149)
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Discussion

Vascular wilt, blight, bakanae disease, etc. caused by Fusarium species have been reported for widespread plant mortality [44,45,46,47]. Selection of disease-resistant plant varieties through in vitro screening has been utilized in the improvement of crops against Fusarium spp, viz. strawberry [30], Musa spp [33], wheat [48, 49], date palm [50], alfalfa plants [51], and passion fruit [52].

This study is the first report of in vitro screening and selection of resistant D. sissoo genotypes against Fsd vascular wilt. Among ten genotypes, it can be concluded that the callus of genotype 14 has resisted the fungal infection, followed by genotype 66. But in genotype 66, the resistance diminished gradually with an increase in conidial concentration to 1 × 105 conidial/ml. Similar studies of callus-fungal interactions for disease resistance selection have been reported in woody species like Acacia pulchella, Eucalyptus calophylla, E. marginata [31], Pinus eschinata and P. virginiana [53], Prunus persica [54], Pinus ellottii [55], Citrus sinensis and C. limon [56], Fagus sylvatica [57], Pinus nigra and P. sylvestris [58], and Malus domestica [59].

In vitro cloned plantlets of D. sissoo genotypes infected under in vitro condition with conidial suspension of Fsd showed results similar to callus. After infection, it was observed that fungal mycelium grew rapidly and a cottony mass of mycelium could be seen around the rhizosphere of plantlets, which may be due to humid conditions of the culture vessels providing a favorable environment for mycelial growth [60]. Nonetheless, on the 15th day of observation, plantlets of genotypes 14 and 66 had a significantly lesser disease severity index (between 2 to 3) implying that initial symptoms of wilt appeared after fungal growth around the rhizosphere but the plantlets remained green, healthy, and had fewer necrotic lesions in the roots whereas plantlets of remaining eight genotypes completely wilted and died. Similar findings have been reported for other plantlet-microbe interactions [33, 51, 53, 61,62,63,64,65].

In vitro screening of clonal host genotypes against specific strains of the pathogen in a dual culture setup is a perfect system for gnotobiotic studies in plant-microbe interaction and it gives an opportunity to estimate the resistance or susceptibility of clones of host plant (D. sissoo genotypes) by ensuring only one microbe (Fsd isolate in this case) is infecting only one host genotype in a culture flask in axenic condition with growth environment. Plantlets with complete root and shoot system or callus (mostly representing the unorganized cellular growth) both of the same genotype showed a similar trend. Growth condition and nutrient rich MS growing media favor the growth of microbe rather than the host in this dual culture setup and thus ensuring strict criteria for selection of resistant genotypes.

Conclusions

The study, thus, concludes, that callus of two genotypes of D. sissoo (14 and 66) showed resistance against Fsd under in vitro conditions whereas the remaining eight genotypes were susceptible. Similar results were observed for in vitro screening of plantlets of D. sissoo genotypes against Fsd. This suggests that in vitro screening of candidate genotypes of D. sissoo against Fsd under gnotobiotic conditions may be an effective as well as a quick method for screening and selection of disease-resistant genotypes. Moreover, by this method, a large number of D. sissoo genotypes could be screened in limited time and space, hence, assisting in the process of screening and selection of disease-resistant genotypes.