In vitro selection of vanilla plants resistant to Fusarium oxysporum f. sp. vanillae

Abstract

Fusarium oxysporum f. sp. vanillae (Fov), the most devastating pathogen of Vanilla planifolia, an important orchid used in the food industry, causes stem and root rot. Vanilla genotypes that are resistant to Fov currently do not exist, so vanilla plants that are resistant to this fungus are needed. In vitro selection offers an effective means to accomplish this objective. Resistant shoots were selected on Murashige and Skoog medium (MS) containing 8.88 µM of 6-benzyladenine and various concentrations of Fov culture filtrate (30, 40 and 50%, v/v). The control treatment (0%) was exclusively MS medium without any culture filtrate. After two 60-day selection cycles, 40 resistant shoots were obtained. In vitro, about 35% of the shoots were resistant to 50% Fov culture filtrate, while in vivo tests indicated that 26.6% of the plants had acquired resistance to the pathogen under greenhouse conditions after 9 weeks. The protocol employed in this study, which forms part of a wider genetic improvement program for this orchid, allowed Fov-resistant V. planifolia plants to be obtained within 420 days.

Full text

1 23
Acta Physiologiae Plantarum
ISSN 0137-5881
Volume 41
Number 3
Acta Physiol Plant (2019) 41:1-8
DOI 10.1007/s11738-019-2832-y
In vitro selection of vanilla plants resistant
to Fusarium oxysporum f. sp. vanillae
Marco A.Ramírez-Mosqueda, Lourdes
G.Iglesias-Andreu, Jaime A.Teixeira da
Silva, Mauricio Luna-Rodríguez, Juan
C.Noa-Carrazana, et al.

1 23
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Vol.:(0123456789)
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Acta Physiologiae Plantarum (2019) 41:40
https://doi.org/10.1007/s11738-019-2832-y
ORIGINAL ARTICLE
In vitro selection ofvanilla plants resistant toFusarium oxysporum f.
sp. vanillae
MarcoA.Ramírez‑Mosqueda1,2· LourdesG.Iglesias‑Andreu3· JaimeA.TeixeiradaSilva4·
MauricioLuna‑Rodríguez5· JuanC.Noa‑Carrazana3· JoséR.Bautista‑Aguilar3· OttoR.Leyva‑Ovalle1·
JoaquínMurguía‑González1
Received: 20 June 2018 / Revised: 18 February 2019 / Accepted: 25 February 2019
© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019
Abstract
Fusarium oxysporum f. sp. vanillae (Fov), the most devastating pathogen of Vanilla planifolia, an important orchid used
in the food industry, causes stem and root rot. Vanilla genotypes that are resistant to Fov currently do not exist, so vanilla
plants that are resistant to this fungus are needed. Invitro selection offers an effective means to accomplish this objective.
Resistant shoots were selected on Murashige and Skoog medium (MS) containing 8.88µM of 6-benzyladenine and various
concentrations of Fov culture filtrate (30, 40 and 50%, v/v). The control treatment (0%) was exclusively MS medium without
any culture filtrate. After two 60-day selection cycles, 40 resistant shoots were obtained. Invitro, about 35% of the shoots
were resistant to 50% Fov culture filtrate, while invivo tests indicated that 26.6% of the plants had acquired resistance to
the pathogen under greenhouse conditions after 9 weeks. The protocol employed in this study, which forms part of a wider
genetic improvement program for this orchid, allowed Fov-resistant V. planifolia plants to be obtained within 420 days.
Keywords Fusarium culture filtrate· Resistance screening· Vanilla stem and root rot· Vanilla planifolia
Introduction
Vanilla (Vanilla planifolia Jacks.) is an economically valu-
able orchid since its fruits contain an organic compound,
vanillin, which has considerable value in several industries
(Ramírez-Mosqueda and Iglesias-Andreu 2015). This orchid
has limited genetic variability as a result of asexual propa-
gation, making it susceptible to diseases such as Fusarium
oxysporum f. sp. vanillae (Fov), which causes soil-borne
stem and root rot (Pinaria etal. 2010; Ramírez-Mosqueda
etal. 2015). Fov can survive in soil for as long as 30years
(Ploetz 2006) and is capable of infecting vanilla plants after
4 months if they are planted in Fov-infested soil, causing a
reduction in growth and productivity (Pinaria etal. 2010;
Hernández-Hernández 2011).
Fov spreads quickly in favorable conditions such as
high humidity, high density in plantations and the disease
incidence of cultivated plants, reducing the effectiveness
of fungicides (He 2007; Hernández-Hernández 2011). In
addition, the excessive use of fungicides in vanilla crop-
ping systems disrupts the life cycle of native mycorrhizal
fungi which in turn alters the ability of vanilla plants to
absorb nutrients, thereby severely affecting the production
Communicated by E. Kuzniak-Gebarowska.
* Marco A. Ramírez-Mosqueda
marcoa.rm.07@gmail.com
* Lourdes G. Iglesias-Andreu
liglesias@uv.mx
1 Laboratorio de Cultivo de Tejidos Vegetales, Colegio de
Postgraduados Campus Córdoba, Km. 348 de la Carretera
Federal Córdoba-Veracruz, Congregación Manuel León,
Amatlán de los Reyes, Veracruz, Mexico
2 Laboratorio de Micropropagación Vegetal, Facultad
de Ciencias Biológicas y Agropecuarias Región
Córdoba-Orizaba, Universidad Veracruzana, Amatlan de los
Reyes, 94945Veracruz, Mexico
3 Laboratorio de Cultivo de Tejidos Vegetales, Instituto
de Biotecnología y Ecología Aplicada (INBIOTECA),
Universidad Veracruzana, Av. de las Culturas Veracruzanas
No. 101, Campus para la Cultura, las Artes y el Deporte,
Col. Emiliano Zapata, 91090Xalapa, Veracruz, Mexico
4 P.O. Box7, Miki-cho post office, Ikenobe, 3011-2,
Kagawa-ken761-0799, Japan
5 Laboratorio de Genética e Interacciones Planta
Microorganismos, Facultad de Ciencias Agrícolas,
Universidad Veracruzana, Circuito Gonzalo Aguirre Beltrán
s/n, Zona Universitaria, 91000Xalapa, Veracruz, Mexico
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Acta Physiologiae Plantarum (2019) 41:40
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(He 2007; Hernández-Hernández 2011). The excessive
use of chemicals is not only eventually harmful to human
health but can also lead to resistance in pathogens that
may be periodically exposed to these compounds (Bosland
1988; He 2007).
In vitro selection can be used to select for disease-
resistant plants using toxic culture filtrates or purified
toxins as a selecting agent (Sharma etal. 2010; Flores
etal. 2012; Mahlanza etal. 2013). Some of these tech-
niques use somaclonal variants with promising characters
that are selected invitro (Larkin and Scowcroft 1981).
Promising genotypes can be selected during developmen-
tal stages like cells, callus, shoots and/or whole plants,
thereby reducing the cycles of selection needed by other
conventional breeding programs (Ravikumar etal. 2007;
Lebeda and Švábová 2010).
In a previous study, we obtained V. planifolia soma-
clonal variants with a high percentage of polymorphism
(71.7%) (Ramírez-Mosqueda and Iglesias-Andreu 2015).
However, the potential of these somaclonal variants has
not been evaluated such as their resistance to Fov. The
aim of this study was to obtain V. planifolia plants that are
resistant to Fov through invitro selection (Fig.1).
Materials andmethods
Fungal culture filtrate
The methodology described by Ramírez-Mosqueda etal.
(2015) was used, with slight edits. A culture filtrate (CF)
was prepared from a pathogenic strain of Fusarium oxyspo-
rum f. sp. vanillae (Fov). This strain, which was named “H3”
(Adame-García etal. 2011), came from the Fov collection of
the Laboratory of Genetics and Plant–Microbe Interactions,
Faculty of Agricultural Sciences, Universidad Veracruzana
(Mexico). A mycelial disc (10mm in diameter) of H3 was
inoculated on Petri dishes carrying potato dextrose agar
(PDA) and incubated in the dark at 26°C for 3 days. The
high pathogenicity of the H3 strain of Fov results from its
high fusaric acid content (Ramírez-Mosqueda etal. 2015).
Mycelial discs were placed in 500mL Erlenmeyer flasks
containing 250mL of potato dextrose broth (Sigma–Aldrich;
pH 6.0). Culture media, which were autoclaved at 121°C
and 124kPa for 15min, were incubated with cultures in the
dark for 3 weeks at 25°C. The fungal culture (FC) was fil-
tered through a nylon net once and then through Whatman®
No. 1 filter paper twice. The FC was refiltered three times
through a re-sterilized continuous Nalgene® filtration system
(Thermo Scientific™, Waltham, MA, USA). Cellulose ester
membranes of different pore sizes (0.45 and 0.22µm) were
used for the filtration. FCs were stored at 5°C in dark bot-
tles. Fresh CFs (no more than 15 days of storage) were used
to perform the invitro selection tests.
Plant material: source, propagation
andmaintenance
To carry out invitro selection, ten shoots of Vanilla planifo-
lia Jacks. (“Mansa” morphotype) that had been regenerated
from friable callus were used. Using the Ramírez-Mosqueda
and Iglesias-Andreu (2015) protocol, this callus was induced
from immature seeds of V. planifolia Mansa morphotype,
and shoots showed considerable somaclonal variation, hav-
ing a high percentage of polymorphism. Shoots were multi-
plied using the method of Ramírez-Mosqueda and Iglesias-
Andreu (2016) in a temporary immersion system (TIB®,
Temporary Immersion Bioreactors; Escalona etal. 1999).
Figure1 provides a flow chart of the methodology used in
this study.
In vitro selection ofV. planifolia shoots resistant
tohigh concentrations ofFov: first cycle ofselection
Once the required amount of plant material was obtained
(i.e., 120 shoots), invitro selection was carried out by
Fig. 1 Flow chart for in vitro selection of Vanilla planifolia plants
resistant to culture filtrates of Fusarium oxysporum f. sp. vanillae.
BA 6-benzyladenine, CF culture filtrate, MS Murashige and Skoog
medium, TDZ thidiazuron
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Acta Physiologiae Plantarum (2019) 41:40
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diluting the CF obtained from the Fov H3 strain to concen-
trations exceeding the median lethal dose (LD50) (30% v/v).
In this context, Ramírez-Mosqueda etal. (2015) determined
that 30% (v/v) FC reduced the survival of a population of
susceptible shoots by 50%. A total of 120 shoots (30 shoots
per treatment) 2–3cm long were cultured in semisolid
Murashige and Skoog (1962) (MS) medium supplemented
with 8.88µM 6-benzyladenine (BA) and 30g L−1 sucrose.
Medium pH was adjusted to 5.8 ± 0.2 and solidified with
2.5g L−1 Gelrite™ (Sigma-Aldrich) before autoclaving at
121°C and 124kPa for 15min. Subsequently, different CF
concentrations (0, 30, 40 and 50% v/v, based on the total MS
medium used) were added on a laminar flow bench. Fresh
CF was added to MS culture medium through a 0.22µm
syringe filter (Millex®, Darmstadt, Germany). The control
treatment was MS medium without CF. Cultures were incu-
bated at 25 ± 2°C, a 16-h photoperiod and a photosynthetic
photon flux density of 50µmol m−2s−1 provided by fluores-
cent lamps (60W Osram®, Munich, Germany). The percent-
age survival of shoots and symptoms, which were assessed
visually, was evaluated after 8 weeks of culture. The visual
scale included three symptoms: none, a necrotic base and
necrotic or dead shoots.
Micropropagation androoting ofselected shoots
Ten shoots that were resistant to 50% (v/v) CF were selected
after 8 weeks (i.e., shoots that showed least symptoms).
These shoots were referred to as RH350 (i.e., resistant to
50% CF of the Fov H3 strain). These shoots were micropro-
pagated and rooted using the method of Ramírez-Mosqueda
and Iglesias-Andreu (2016) in a TIB® (Escalona etal. 1999).
For rooting, half-strength MS medium free of any plant
growth regulators but supplemented with 30g L−1 sucrose
was used. Immersion frequency was 2min every 4h and
utilized an average of 25mL explant−1. Cultures were incu-
bated in identical conditions described above. The control
treatment consisted of micropropagated and rooted shoots
that did not derive from invitro selection and that were not
treated with Fov CF. The number of shoots per explant,
percentage rooting, and number and length of roots were
evaluated.
In vitro selection ofV. planifolia plantlets resistant
tohigh concentrations ofFov: second cycle
ofselection
One hundred RH330 rooted invitro plantlets (6–8cm
length) were exposed for the second time invitro to 50%
(v/v) CF in semisolid MS medium, as described above for
the invitro selection protocol under identical culture condi-
tions, with the same CF-free control. Shoot survival (%) was
evaluated after 60 days.
Acclimatization ofselected RH350 plants
As described similarly in Ramírez-Mosqueda etal. (2015),
surviving plants (40 plants, 8–10cm long) were rinsed with
tap water and planted in a 1:1 (v/v) mixture of peat moss
(Premier, Rivière-du-Loup, Canada) and Agrolita® (glassy
volcanic rock) (Agrolita, Tlalnepantla de Baz, Mexico) in
50 × 30 × 5cm trays. Plantlets were kept in a greenhouse
under the following conditions: natural light, 50% shade
(Mallan®), 80–95% relative humidity, 28–32°C. Nitrofoska®
(N, 25; P, 10; K, 17) (PS, COMPO, Zapopan, Mexico) was
applied as a foliar fertilizer once a week, and plantlets were
watered three times a week. When plants reached 30cm
after 8 weeks, they were transferred to separate individual
containers (5 × 5 × 25cm) using the same substrate. After
8 weeks of culture under these conditions, the percentage
survival of acclimatized plantlets was determined.
Testing invivo resistance ofregenerated plants
toFov: third cycle ofselection
This experiment was conducted following the methodology
proposed by Thakur etal. (2002). A total of 30 selected
RH350 plants (resistant to 50% Fov CF, 9 weeks old and
25–30cm long) and 30 control plants were infected invivo
by adding 10mL of a freshly prepared Fov mycelial suspen-
sion around each plant. To check the stability of resistance,
plant symptoms were observed. After 4 weeks, plant sur-
vival was assessed while disease severity was graded based
on visual observations according to a 0–4 scale proposed
by Baayen and de Maat (1987) that was originally devel-
oped for carnation. In this scale, as described by Thakur
etal. (2002), 0 indicates no symptoms (resistant), 1 indicates
slight symptoms (one wilted leaf at the stem base or a brown
discoloration of the stem base surface; resistant), 2 indicates
a well-developed characteristic unilateral wilt of otherwise
still healthy plants (moderately resistant), 3 indicates severe
wilt (susceptible) and 4 indicates complete wilt or death
(highly susceptible).
Statistical analysis
All experiments were performed in duplicate using a com-
pletely randomized design (Gomez and Gomez 1983). For
the invitro analysis, to serve as a replicate, 30 shoots per
treatment were used and two shoots were placed in a bottle
containing 20mL of medium. For the invitro analysis, 30
selected plants and 30 non-selected plants (control) were
used for this analysis. Only the data obtained for all the
invitro shoot experiments were analyzed by analysis of
variance (ANOVA) followed by Tukey’s multiple range
test (p ≤ 0.05). For the other experiments, both invitro
and invivo, the Mann–Whitney U test (p ≤ 0.05) was used.
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SPSS software (version 21 for Windows Inc., Chicago, IL,
USA) was used for both statistical tests. Also using SPSS
software, data were tested for normality and variance
homogeneity using the Kolmogorov–Smirnov and Lev-
ene tests, respectively. Variables that did not meet these
assumptions were log transformed using the natural loga-
rithm (ln), and all percent data were arc-sin transformed.
Results
In vitro selection ofV. planifolia shoots resistant
tohigh concentrations ofFov: first cycle ofselection
Our results show that the survival of V. planifolia shoots
decreased as CF concentration in the culture medium
increased (Table1). Lowest survival (35.7%) was observed
with 50% CF while 100% shoot survival was observed in
the control treatment. All shoots exposed to CF displayed
the same symptom (necrotic base) regardless of the CF
concentration (Fig.2) while 35.7% of the shoots (from
indirect organogenesis) were resistant to 50% CF (Fig.2).
These shoots were referred to as RH350 (i.e., resistant to
50% CF of the Fov H3 strain).
Micropropagation androoting ofselected shoot
material
Multiple RH350 shoots developed after 4 weeks of culture in
the BIT® (Table2; Fig.3a), forming 13.20 ± 0.96 shoots per
explant. After 4 weeks of culture in BIT® (Table2; Fig.3b),
shoots formed an average of 3.7 ± 0.9 roots per shoot. Aver-
age root length was 2.1 ± 0.9cm (Table2).
In vitro selection ofV. planifolia plantlets resistant
tohigh concentrations ofFov: second cycle
ofselection andacclimatization ofselected plant
material
After 8 weeks of a second exposure invitro of RH350 plant-
lets to 50% CF, a higher number (40.0 ± 3.2%) of plants
Table 1 In vitro selection of V. planifolia shoots resistant to high con-
centrations of Fov: first cycle of selection
Numbers represent mean ± SE (standard error). n = 240 plants
CF culture filtrate
Means with different letters are significantly different (Tukey’s multi-
ple range test, p ≤ 0.05)
Fungal filtrate concentration Survival (%) Symptomatology
Control without CF 100.0 ± 0.0 a None
30% of CF 60.0 ± 0.7 a Necrotic base
40% of CF 53.3 ± 0.6 a Necrotic base
50% of CF 35.7 ± 0.8 b Necrotic (dead) shoots
Fig. 2 In vitro selection of
Vanilla planifolia shoots resist-
ant to culture filtrate (CF) of
Fusarium oxysporum f. sp.
vanilla cultured in MS medium
supplemented with 8.88µM BA
and 30g L−1 sucrose. From left
to right, 0%: control without
CF; 30–50%: dilutions of the
fungal CF (30, 40 and 50%),
respectively
Table 2 In vitro selection of V. planifolia plantlets resistant to high
concentrations of Fov: second cycle of selection
Numbers represent mean ± SE (standard error)
CF culture filtrate
Means with different letters are significantly different (Mann–
Whitney U test, p ≤ 0.05). % data were arc-sin transformed. Con-
trol = plants derived from the medium without Fov FC
Treatments Symptomatology
Micropropagation (shoots per explant)
Control 13.2 ± 0.9 a None
RH350 vs CF 50% 12.7 ± 0.3 a None
Rooting (%)
Control 100.0 ± 0.0 a None
RH350 vs CF 50% 99.0 ± 0.7 a None
Rooting (roots per shoot)
Control 3.7 ± 0.9 a None
RH350 vs CF 50% 3.5 ± 0.8 a None
Rooting (length of the roots)
Control 2.1 ± 0.9 a None
RH350 vs CF 50% 2.2 ± 0.7 a None
In vitro selection of plantlets (survival %)
Control 100.0 ± 0.0 a None
RH350 vs CF 50% 40.0 ± 3.2 b Necrotic base
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were resistant to 50% CF compared to the control (Table2;
Fig.3c). However, necrotic and/or dead shoots were still
observed (Fig.3c). Surviving plants (8–10cm length)
were acclimatized in a greenhouse, and 90.0% survival was
achieved 8 weeks after invitro RH350 plantlets were trans-
ferred to the greenhouse (Figs.3d, 4).
In vivo resistance testing ofregenerated plants
againstFov: third cycle ofselection
Eight-week-old RH350 plants derived from 50% CF treat-
ment showed higher resistance (26.6 ± 2.8%) to Fov than
control plants (Table2; Fig.5). However, in the selected
plants, different levels of damage were observed (Table3;
Fig.5). Several (20.0%) resistant plants showed no symp-
toms (group 0 = resistant; Table3; Fig.5a), while some
(6.6%) showed vigorous growth and were slightly affected
(group 1 = resistant; Table3; Fig.5b). Moderately resist-
ant plants (6.6%) showed partial yellowing (group 2 = mod-
erately resistant; Table3; Fig.5c). However, some plants
(6.6%) obtained after in vitro selection were either still
susceptible and showed severe wilt (group 3 = susceptible;
Table3; Fig.5d) or were highly susceptible (60.0%), show-
ing complete wilt (group 4 = highly susceptible; Table3;
Fig.5e). About one quarter (26.6%) of plants derived from
invitro selection (Fig.2) showed resistance to Fov after
invitro tests (Table3).
Discussion
In this study, V. planifolia plants resistant to disease caused
by Fov were selected to help mitigate stem and root rot in
this orchid. The invitro selection of plants resistant to dis-
eases caused by various pathogens has been reported for
different plant species, including sugarcane (Fusarium
sacchari) (Mahlanza etal. 2013), pea (F. solani) (Švábová
etal. 2011), pineapple (F. subglutinans) (Borras and Bermú-
dez 2010), banana (F. oxysporum f. sp. cubense) (Hu etal.
2013), turmeric (F. oxysporum f. sp. zingiberi) (Kuanar etal.
2014), and yellow passion fruit (F. oxysporum f. sp. passi-
florae) (Flores etal. 2012). The majority of these studies are
based on somaclonal variation that occurs during the invitro
culture of plant tissues (Larkin and Scowcroft 1981). How-
ever, some factors increase this variation by different mecha-
nisms, including chemical mutagenesis (Purwati etal. 2007)
or physical mutagenesis (Sharma etal. 2010). Of note, in
our study, somaclonal variation was potentiated by indirect
Fig. 3 In vitro selection of Vanilla planifolia RH350 shoots resist-
ant to culture filtrate (CF) of Fusarium oxysporum f. sp. vanilla, and
greenhouse acclimatization. a Shoots (after 30 days of culture) micro-
propagated in a BIT®; b Rooted shoots (after 30 days of culture) in a
BIT®; c Plantlets (after second cycle of invitro selection, 60 days of
culture) exposed to 50% CF; d Fov-resistant plant 420 days old from
50% CF exposure during acclimatization
Fig. 4 In vivo selection of Vanilla planifolia plants resistant (RH350)
to Fusarium oxysporum f. sp. vanillae, plant from the in vitro selec-
tion process (left) to highly susceptible control plant (right)
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(i.e., callus-induced) organogenesis (Ramírez-Mosqueda and
Iglesias-Andreu 2015).
There are studies of classic genetic improvement of the
Vanilla genus such as those carried out by Koyyappurath
etal. (2015), who evaluated the resistance to Fov of 177
accessions (species and interspecific hybrids) in the field,
but found that only 30 had some degree of resistance, i.e.,
16.9%. In contrast, in invitro tests, from a total of 103 evalu-
ated accessions, 19 had some degree of resistance to Fov,
i.e., 18.4%. In their study, long-term (4-year) field evalu-
ations were carried out from 2009 to 2013, without taking
into account the years required for the development of inter-
specific crosses. In contrast, in our study, a higher percent-
age (26.6%) of Fov-resistant plants was obtained through an
invitro-to-ex vitro selection cycle that lasted only 420days.
The invitro plantlets resistant to 50% (v/v) CF obtained
in our study are of great interest to try to mitigate stem
and root disease in V. planifolia. Ramírez-Mosqueda etal.
(2015) showed that V. planifolia plants that do not originate
from direct organogenesis, and with a high degree of poly-
morphism, were susceptible to 30% Fov CF. Kuanar etal.
(2014) obtained Curcuma longa cv. ‘Suroma’ plants that
were resistant to F. oxysporum f. sp. zingiberi by adding a
7% (v/v) CF to the culture medium. For the invitro selection
of Passiflora edulis, Flores etal. (2012) used a 14% CF of
filtered F. oxysporum f. sp. passiflorae.
In our study, similar percentages of Fov-resistant plants
were observed throughout the process described in Fig.1
using CFs. Fov resistance of 35.7% was obtained during the
first cycle of selection of shoots invitro, 40.0% in the sec-
ond cycle of invitro selection in plants, and 26.6% in more
developed plants in the invivo tests. In most selection stud-
ies, there is a relationship between invivo and invitro resist-
ance of plants selected (Kuanar etal. 2014; Mahlanza etal.
2013; Hu etal. 2013; Flores etal. 2012; Koyyappurath etal.
2015). It is possible to implement plant–pathogen interac-
tions invitro because the plants grown in this system grow in
an environment that mimics field conditions (Predieri 2001).
In this study, we demonstrated the usefulness of using
a fungal filtrate strain “H3” from a pathogenic strain of
Fov in a V. planifolia invitro selection program. In this
regard, CFs contain other phytotoxic compounds that may
result in the selection of plants resistant to other aspects
except for the target pathogen (Remotti etal. 1997). Some
authors used the purified toxin fusaric acid to select plants
invitro resistant to fungi: Flores etal. (2012) found plants
Fig. 5 In vivo selection in
greenhouse of Vanilla planifolia
plants resistant to Fusarium
oxysporum f. sp. vanillae
according to the scale of Baayen
and De Maat (1987). a Resist-
ant plants (group 0) without
symptoms; b resistant plants
(group 1) with slight symp-
toms; c moderately resistant
plants (group 2) showed partial
yellowing; d susceptible plants
(group 3) with severe wilt; e
highly susceptible plants (group
4) with complete wilt
Table 3 In vivo resistance
testing of regenerated plants
against Fov: third cycle of
selection
Numbers represent mean ± SE (standard error). % data were arc-sin transformed. 0 Control = plants derived
from the medium without Fov FC
In the in vivo tests, the control treatment consisted of plants of Vanilla planifolia susceptible to Fov
0 = resistant; 1 = resistant; 2 = moderately resistant; 3 = susceptible; 4 = highly susceptible (Baayen and de
Maat 1987)
Means with different letters are significantly different (Mann–Whitney U test, p ≤ 0.05).
Plants No. inocu-
lated plants
Groups Total survival (%) Resistant plants (%)
01234
Control 30 0 0 2 8 20 0.0 ± 0.0 b 0.0 ± 0.0 b
Selected plants
(RH350)
30 6 2 2 2 18 40.0 ± 2.8 a 26.6 ± 2.8 a
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resistant to Colletotrichum spp. while Remotti etal. (1997)
found plants resistant to F. oxysporum f. sp. gladioli. How-
ever, fusaric acid is not a selective toxin, so other com-
pounds in the CF might be necessary for its selectivity
(Borras and Bermúdez 2010).
Under greenhouse conditions, 26.6% of vanilla plants
were resistant to Fov. A similar percentage of resistant
plants was reported in other studies involving invitro
selection (Flores etal. 2012; Hu etal. 2013; Mahlanza
etal. 2013; Kuanar etal. 2014).
Conclusions
Somaclonal variants of Vanilla planifolia resistant to Fov
were obtained through invitro culture and subsequent
selection. Our results may contribute to a possible solu-
tion for this disease in vanilla, i.e., stem and root rot, and
set a basis for other genetic and biotechnological studies
for this orchid genus.
Limitations ofthestudy andfuture perspectives
The study has several limitations. Only duplicate experi-
ments were performed due to the need for a large number
of plants, so replicate studies with larger sample sizes
would be required in the future. Root-based phytopatho-
logical observations will be required since only shoot-
based observations were made in this study. Molecular
analyses to fortify the presence of Fov in invitro, green-
house and field plant material, and to provide additional
proof of resistance to Fov, will be required in future exper-
iments. The heritability of Fov resistance in the M2 and
subsequent generations needs to be tested.
Author contribution statement LGIA, MARM and JATS
conceived and designed the research. MARM and JRBA
conducted all experiments. MARM, MLR, and JCNC ana-
lyzed and reviewed the statistical analysis. MARM, LGIA
and JATS wrote the manuscript. LGIA, MARM, JCNC,
JATS, MLR and JRBA read, edited and approved the
manuscript.
Acknowledgements The authors thank the “Programa para el Desar-
rollo Profesional Docente (PRODEP)” for financial support provided
for the project “Biotechnological Basis for the Genetic Improvement of
Vanilla planifolia” within the “Conservation, Management and Plant
Breeding Network”. MARM thanks the Consejo Nacional de Ciencia
y Tecnología (CONACyT) for the grant scholarship No. 275736, which
allowed this work to be performed.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicting
interests.
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