Chauhan et al. Journal of Genetic Engineering and Biotechnology
https://doi.org/10.1186/s43141-021-00132-3
(2021) 19:35
Journal of Genetic Engineering
and Biotechnology
RESEARCH
Open Access
Gnotobiotic evaluation of Dalbergia sissoo
genotypes for resistance against Fusarium
solani via dual culture set up
Monika Chauhan1, Ajay Thakur1* , Yashaswi Singh1 , M. Maqbool Rather2 and Nirmal S. K. Harsh3
Abstract
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.
Keywords: Fusarium solani f. sp. dalbergiae, Fusarium wilt, Genetic variation, Gnotobiota, In vitro screening,
Shisham mortality, Plant-microbe interaction
* Correspondence: mithoojorhat@yahoo.co.in; thakura@icfre.org
1
Tissue Culture Laboratory, Genetics and Tree Improvement Division, Forest
Research Institute, Dehra Dun, Uttarakhand 248006, India
Full list of author information is available at the end of the article
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
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Chauhan et al. Journal of Genetic Engineering and Biotechnology
Page 2 of 9
(2021) 19:35
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 [1–3]. 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].
Widespread mortality in D. sissoo (Fig. 1) has been reported from many parts of India [7–14] 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.
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 [30–36]. 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
Table 1 Source location of selected genotypes of D. sissoo in
the study
Fig. 1 D. sissoo completely wilted (a) and healthy (b) trees in their
natural habitat (riverine forest) Thano forest range, Dehra Dun, India
Sl. Genotype
No. number
Source location
Locality
District
State
1.
10
Sabalgarh, Pathri
Haridwar
Uttarakhand
2.
14
Sabalgarh, Pathri
Haridwar
Uttarakhand
3.
19
Shah Mansurpur
Saharanpur
Uttar Pradesh
4.
24
Collectorbuck Ganj
Bareilly
Uttar Pradesh
5.
41
Hasanpur, Tulsipur
Gonda
Uttar Pradesh
6.
66
Chhachhrauli Range Yamuna Nagar Haryana
7.
201
Hasanpur, Tulsipur
Gonda
Uttar Pradesh
8.
204
Tulsipur
Gonda
Uttar Pradesh
9.
232
Birpur-Bhambar
Gonda
Uttar Pradesh
10.
237
Bankatwa
Gonda
Uttar Pradesh
Chauhan et al. Journal of Genetic Engineering and Biotechnology
(2021) 19:35
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].
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 × 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.
Page 3 of 9
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 =
Chauhan et al. Journal of Genetic Engineering and Biotechnology
Page 4 of 9
(2021) 19:35
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). Nonnormal 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.
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
Table 2 Effect of conidial concentration of Fsd (1145 and 1149) on callus of D. sissoo genotypes
Genotypes
Spore concentration
1 × 101
1 × 103
1 × 105
Mean (cm)
1 × 101
Fungal diameter for isolate 1145 (cm)
1 × 103
1 × 105
Mean (cm)
Fungal diameter for isolate 1149 (cm)
14
0.8
0.8
1.1
0.9
1.1
1.1
1.3
1.2
66
0.9
0.9
1.3
1
1.1
1.1
1.5
1.2
19
0.9
1.3
2.4
1.5
1.2
1.6
2.6
1.8
41
1.0
1.9
3.0
2.9
1.2
2.0
3.0
2.1
24
1.0
1.8
3.4
2.1
1.3
2.0
3.6
2.3
10
1.1
2.2
3.9
2.4
1.4
2.3
3.8
2.5
201
0.9
1.1
2.2
1.4
1.2
1.5
2.6
1.7
204
1.2
2.1
4.2
2.5
1.4
2.3
4.0
2.6
232
1.4
2.4
5.0
2.9
1.5
2.5
4.7
2.9
2.8
2.8
237
1.3
2.3
4.78
Mean (cm)
1.04
1.68
3.12
1.5
2.4
4.5
1.28
1.88
3.14
Variable
LSD at 5%
Variable
LSD at 5%
Genotypes
0.20
Genotypes
0.13
Treatment
0.29
Treatment
0.17
Genotypes × treatment
0.34
Genotypes × treatment
0.22
Chauhan et al. Journal of Genetic Engineering and Biotechnology
(2021) 19:35
Page 5 of 9
Fig. 2 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
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
Chauhan et al. Journal of Genetic Engineering and Biotechnology
(2021) 19:35
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).
Discussion
Vascular wilt, blight, bakanae disease, etc. caused by Fusarium species have been reported for widespread plant
mortality [44–47]. 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
Page 6 of 9
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, 61–65].
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.
Abbreviations
PDA: Potato dextrose agar; MS: Murashige and Skoog basal media; BAP: 6Benzylaminopurine; NAA: 1-Naphthaleneacetic acid; IBA: Indole-3-butyric
acid; 2,4-D: 2,4-Dichlorophenoxyacetic acid; CMC: Carboxymethyl cellulose;
Fsd: Fusarium solani f. sp. dalbergiae; CRD: Completely randomized design;
ANOVA: Analysis of variance; LSD: Least significant difference; KW: KruskalWallis test
Acknowledgements
Authors acknowledge Dr. Ashok Kumar, Genetics and Tree Improvement
Division, FRI, Dehra Dun for providing D. sissoo genotypes and Head, Forest
Protection Division, Forest National Type Culture Collection, FRI, Dehra Dun,
India, for providing the fungal strains. The authors thank the Director and
Vice Chancellor, FRI Deemed University, Dehra Dun, India, for providing
facilities to conduct the study.
Authors’ contributions
MC and AT conceived and designed the research on in vitro studies. NSKH
helped in fungal studies. MC performed the experiments. AT, MC and YS
analyzed the results. MC, YS and MMR wrote the manuscript with assistance
from AT and NSKH. All authors have read and agreed to the published
version of the manuscript.
5th day
Isolate no.
7th day
1145
1149
9th day
1145
1149
11th day
1145
1149
1145
13th day
1149
1145
15th day
1149
1145
1149
Genotype no. Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb Meana Rankb
1
6
1.8
5
1.8
7.5
2.6
5
2.8
5.5
3.8
6
3.8
6.0
4.8
6
5.0
7.5
5
7
5.0
6.5
5
6.5
14
0.4
1
0.4
1
0.6
1.0
1
2
1.0
1.0
1.6
1
1.6
1.0
1.8
1
2.2
1.0
1.8
1
2.4
1.0
2.6
1.0
19
0.8
3.5
0.8
3
1.2
6.0
2
4
1.6
3.0
2.6
3
2.8
3.0
3.8
3
3.6
3.0
4.8
3
5.0
6.5
5
6.5
24
1.2
8.5
2
8
2.0
9.0
3
8
3.0
7.5
3
5
4.0
8.5
5
9
5.0
7.5
5
7
5.0
6.5
5
6.5
41
0.8
3.5
1
4
1.0
4.0
2
4
1.8
4.0
2.8
4
3.0
4.0
4
4
3.8
4.0
5
7
5.0
6.5
5
6.5
66
0.6
2
0.6
2
0.8
2.0
1
2
1.2
2.0
2
2
1.8
2.0
2.4
2
2.6
2.0
2.6
2
2.8
2.0
2.8
2.0
201
1.0
6
2
8
1.0
4.0
3
8
2.8
5.5
4
8
3.8
6.0
4.8
6
5.0
7.5
5
7
5.0
6.5
5
6.5
204
1.4
10
2
8
1.8
7.5
3
8
3.0
7.5
4
8
3.8
6.0
4.8
6
5.0
7.5
5
7
5.0
6.5
5
6.5
232
1.0
6
2
8
1.0
4.0
3
8
3.2
9.5
4
8
4.0
8.5
5
9
5.0
7.5
5
7
5.0
6.5
5
6.5
237
1.2
8.5
2
8
2.4
10.0
3.2
10
3.2
9.5
4.2
10
4.2
10.0
5
9
5.0
7.5
5
7
5.0
6.5
5
6.5
(2021) 19:35
10
Chauhan et al. Journal of Genetic Engineering and Biotechnology
Table 3 Disease scoring of plantlets of ten D. sissoo genotypes inoculated with Fsd (1145 and 1149)
a
Mean disease score reflects disease severity and brank reflects the ability to resist Fsd infection
Page 7 of 9
Chauhan et al. Journal of Genetic Engineering and Biotechnology
(2021) 19:35
Funding
This research received no external funding.
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Tissue Culture Laboratory, Genetics and Tree Improvement Division, Forest
Research Institute, Dehra Dun, Uttarakhand 248006, India. 2Faculty of
Forestry, SKUAST-Kashmir, Benehama, Ganderbal, Jammu and Kashmir, India.
3
Forest Pathology Division, Forest Research Institute, Dehra Dun, Uttarakhand
248006, India.
Received: 24 September 2020 Accepted: 3 February 2021
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