Gnotobiotic evaluation of Dalbergia sissoo genotypes for resistance against Fusarium solani via dual culture set up

Background Dalbergia sissoo (shisham), an important multipurpose tree native to the Indian subcontinent and also planted in other countries, has been afflicted with large scale mortality in all age groups due to wilt disease, causing huge economic losses. Fusarium solani f. sp. dalbergiae (Fsd) has been identified as one of the causal organisms for wilt disease in D. sissoo. One of the approaches of disease resistance studies involves co-cultivation of trees and pathogens under controlled conditions to screen resistant tree genotypes. A gnotobiotic condition, where the pathogen is known, enables accurate screening of disease-resistant genotypes. In the present study, ten genotypes of D. sissoo were cloned in vitro and evaluated against two strains of Fsd in a dual culture setup under gnotobiotic conditions with an objective to identify resistant genotypes of D. sissoo against Fsd. Results Callus and plantlets of ten genotypes of host plant multiplied in vitro were inoculated with conidial suspension of two strains of Fsd at three concentrations; 1 × 101, 1 × 103, and 1 × 105 conidia/ml. Gnotobiotic evaluation of dual culture setup shows variations among genotypes in their response towards in vitro Fsd infection; and two genotypes (14 and 66) exhibited resistance against Fsd strains. Callus of genotypes 14 and 66 significantly restricted the fungal mycelium growth whereas callus of remaining genotypes was completely infested by Fsd mycelium within 9 days. Similarly, plantlets of genotype 14 and 66 had lesser disease severity and remained green and had fewer necrotic lesions in roots whereas plantlets of the remaining eight genotypes died within 15 days. Conclusion Gnotobiotic evaluation of callus and plantlets of ten genotypes of D. sissoo against Fsd strains has reduced time and space otherwise required for field trials. Genetic variations amongst the genotypes resulted in varying responses towards virulent Fsd strains and only two out of ten genotypes showed promising resistant characteristics. In dual culture setup, both callus and plantlets of the same genotypes responded similarly against Fsd strains, which signify that in vitro screening can be used as an indirect selection method for disease resistance.


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 wheatbased agroforestry system as it is deciduous and fixes nitrogen [5,6].
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 diseaseresistant 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 cocultured 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].

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].

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, bestresponded 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 × 10 5 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/m 2 /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 × 10 1 , 1 × 10 3 , and 1 × 10 5 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 × 10 1 , 1 × 10 3 and 1 × 10 5 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 × 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.

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 × 10 5 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 ( 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 × 10 5 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 × 10 1 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 × 10 1 to 1× 10 5 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 × 10 1 to 1 × 10 5 conidia/ml. However, for isolate 1149 an increase in conidial concentration from 1 × 10 1 conidia/ml to 1 × 10 5 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 × 10 1 to 1 × 10 3 conidia/ml as well as from 1× 10 3 conidia/ml to 1× 10 5 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 × 10 1 to 1 × 10 3 conidia/ml, however fungal spread on callus differed significantly when conidial concentration of both isolates was increased from 1 × 10 3 conidia/ml to 1 × 10 5 conidia/ml. In other genotypes spread on callus differed significantly when conidial concentration of both isolates was increased from 1 × 10 1 to 1 × 10 3 conidia/ml as well as from 1 × 10 3 to 1× 10 5 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 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).
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 plantletmicrobe 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 diseaseresistant 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.