In vitro Selection for Wilt Resistance in Guava (Psidium guajava
L.) cv. Allahabad Safeda
In vitro selection is a feasible method for developing wilt resistant or tolerant genotypes of guava. Unlike other crops, this approach has not been well established for guava. In vitro selection system against Fusarium wilt was employed with the aim to establish a recurrent selection system in guava (Psidium guajava L.) both at the cellular and plant level. In vitro somatic embryogenesis derived plantlets of guava cv. Allahabad Safeda were screened against Fusarium sp. culture filtrate at various concentrations of (0, 5, 25, 50 and 100%, v/v) supplemented in Murashige and Skoog (MS) basal liquid medium up to four selection cycles. The effect of Fusarium culture filtrate on plantlet survival and proliferation were observed to be directly related to the concentration in the selection media. The current study revealed that in vitro screening of guava plantlets using Fusarium sp. culture filtrate when exposed to the ten different culture filtrates at five concentrations of which; two of the culture filtrates of Fusarium solani (F2 and F15) shows maximum mortality of guava plantlets at 50% culture filtrate and later shows some level of tolerance to the culture filtrates. However, two of the culture filtrates F18 and F24 of Fusarium oxysporum f. sp. psidii shows maximum regeneration i.e., up to 100% even after fourth selection cycle. Current study revealed that Fusarium sp. culture filtrate can be potentially employed as a potent selection agent for carrying out in vitro selection approach against wilt disease of guava. The successful recurrent selection system developed in vitro on the basis of current research can probably help in ameliorate the problem of wilt in guava.
Received: March 31, 2012;
Accepted: May 18, 2012;
Published: July 03, 2012
Guava (Psidium guajava L.) belongs to the family Myrtaceae which has
more than 80 genera and 3,000 species distributed throughout the tropics and
subtropics (Chandra et al., 2010a). It is regarded
as nutritionally valuable and remunerative crop used for both, table and processing
purposes. It is liked by fruit growers due to its wide adaptability and higher
return per unit area. But its successful cultivation is hampered by a number
of biotic and abiotic factors. Among the biotic factors, diseases take a heavy
toll. Production of guava has been severely affected worldwide by a soil borne
disease called wilt. The disease was first reported in India by Das
Gupta and Rai (1947). Wilt of guava is one of the most devastating problems
faced by the orchardists in India, as the disease is totally uncontrolled through
chemical or physical measures (Misra, 2006). Control
measure other than eradication of diseased trees, were not fully successful.
Although several fungal pathogens have been reported as the causal agents of
wilt in guava by different workers but Fusarium oxysporum f. sp. psidii
and Fusarium solani were found to be the most important pathogen associated
with wilt (Misra, 2006; Misra and
Gupta, 2007; Gupta et al., 2010). However,
Fusarium solani is the most virulent and pathogenic fungi in nature most
commonly found wherever the guava wilt disease is to be reported (Gupta
et al., 2010). First external symptoms of disease are the appearance
of yellow coloration with slight curling of leaves of the terminal branches.
At later stage, plants show unthriftiness with yellow to reddish discolouration
of the leaves and subsequently its premature shedding. Fruits of all the affected
branches remain underdeveloped, hard and stony (Misra, 2006).
Later on, the entire plant defoliates and dies. Mainly two types of wilt symptoms
are identified-slow wilt and sudden wilt (Chakraborty and
Singh, 1989). Substantial research work has been done on wilt disease of
guava regarding chemical and biological controls through botanicals and biocontrol
agents but, no workable solution has been successful yet. Wilt infected mother
plants of guava are playing major role in spreading the disease beyond leaps
and bounds. Therefore, it is of the utmost importance to develop wilt resistant
guava cultivar in days to come otherwise the guava industry may collapse (Chandra
and Mishra, 2007; Chandra et al., 2010a).
Development of resistant varieties is thought to be the most viable strategy
to overcome this problem. Selection for disease resistance breeding in fruit
crops is an alternative to genetic engineering (Jayasankar
and Litz, 1998; Hartman et al., 2004; Chandra
and Mishra, 2007; Chandra et al., 2010b).
This in vitro selection approach is easy to use and not encumbered by
intellectual property issues and social concerns currently inhibiting development
of transgenic crops. Thus, it is an attractive adjunct to the already existing
crop improvement strategies through biotechnological interventions. The in
vitro selection approach to select disease resistant/tolerant lines in culture
has been to use phytotoxic fungal culture filtrate or purified toxins produced
by the pathogen (Svabova and Lebeda, 2005; Sengar
et al., 2009). Earlier studies on in vitro selection for disease
resistance trait among the fruit crops viz., peach, lemon, grape, banana and
mango were successfully carried out. These studies suggested the use of in
vitro selection as potential approach for developing enhanced resistance
to phytopathogens. The developmental mechanisms for obtaining disease resistance
through in vitro selection remain obscure. In case of guava; for the
first time a wilt resistant rootstock has been developed successfully using
in vitro cellular selection system in South Africa against Penicillium
sp. (Vos et al., 1998). In India, only a
preliminary study of in vitro selection using fungal culture filtrates
was initiated for the development of a wilt resistant rootstock for Psidium
species (Bajpai et al., 2007) and so far
no reports were available.
The current investigation aims with the objective to explore the effects of Fusarium sp. culture filtrate as a potent selection agent on in vitro regenerated guava plantlets to develop a recurrent selection system in guava which could be of great significance in further development of a wilt resistant genotype.
MATERIALS AND METHODS
The current research investigation was carried out at Biotechnology Laboratory, Central Institute for Subtropical Horticulture, Rehmankhera, Lucknow (U.P.), India during January 2009 to March, 2011.
The immature fruits (70 days after post anthesis) of guava (Psidium guajava
L.) were collected as explant source from guava orchard at C.I.S.H., Rehmankhera,
Lucknow, India. The guava cv. used in the present study was Allahabad Safeda.
Seventy days old immature guava fruits were brought to the laboratory and surface
sterilized with water and then soaked in HgCl2 (0.1%) for 3 min followed
by thorough washing with distilled water in order to remove traces of mercuric
chloride. Under aseptic conditions flame sterilized the fruit using 90% alcohol
and then cut the fruit into two halves and scoops out the immature zygotic embryos
(Akhtar, 2010) embedded in the mesocarp of the fruit
and inoculate into the basal medium.
Culture initiation: The immature zygotic embryos, 70 days or 10 weeks
after post anthesis were used as explant material for inducing somatic embryogenesis
in guava. The zygotic embryos were inoculated in induction medium containing
(Murashige and Skoog, 1962) medium supplemented with 2.0
mg L-1 2, 4-D, 400 mg L-1 L-glutamine, 60 g L-1
sucrose and 0.8% (w/v) agar-agar. The medium prepared adjust pH 5.8 prior to
autoclaving at 121°C at 15 lbs pressure for 20 min. The cultures were incubated
under dark for inducing somatic embryogenesis and thereafter shifted the embryos
to the regeneration media (0.5 mg L-1 BAP and 0.2 mg L-1
NAA) in the light for further regeneration and plantlet development. The plantlets
developed were kept under artificial lightening at 24±2°C for 16/8
h dark. These somatic embryogenesis derived plantlets were now used as explant
source for carrying out in vitro selection.
Pathogen culture: The pathogenic Fusarium sp. isolates were isolated
from the rhizospheric regions of the wilt infected guava plant roots and grown
on potato dextrose agar (PDA, Hi-Media) and incubated at 28±2°C for
7 days and were routinely transferred to medium at every 30 days or 4 weeks.
The morphological and cultural characterizations of the pathogenic Fusarium
sp. culture were compared with those mentioned by Booth (1971).
The details of pathogenic pure cultures of Fusarium solani and Fusarium
oxysporum f. sp. psidii were represented (Table 1).
The pathogenic reference pure Fusarium sp. cultures were confirmed from
Indian Type Culture Collection (ITCC), Division of Mycology and Plant Pathology,
I.A.R.I., New Delhi for identification of Fusarium solani [ITCC No. 5208
(F20) and 5212 (F15)] and Microbial Type Culture Collection and GenBank (Chandigarh,
Punjab) Fusarium oxysporum f. sp. psidii [MTCC No. 3326 (F24)
and 3327 (F30)]. However, Fusarium solani F2, F12 and F20 (Booth,
1971) and Fusarium oxysporum f. sp. psidii F10, F18 and F38
||Fusarium sp. isolates used for in vitro selection
studies against wilt of guava
Preparation of Fusarium sp. culture filtrates: Potato Dextrose Agar (PDA) medium petri dishes were inoculated with Fusarium sp. isolates (Table 1) and incubate under dark at 28°C in BOD incubator. After one week 2 discs of 5 mm diameter (using cork borer) of pathogenic sporulated Fusarium sp. cultures were inoculated into the flasks containing potato dextrose broth under aseptic conditions. Incubate the inoculated broth culture at 28°C in orbital shaker at 120 rpm for 3 weeks or 21 days. Later, the cultures were taken out containing Fusarium sp. culture filtrate is now filtered through four layers of what man No.1 paper and then centrifuged for 10 min at 13000 rpm to remove the traces of mycelium and conidia. After this, the culture filtrate is again filtered through 0.22 μ millipore syringe filter and maintained pH 5.8 before use kept at -20°C before performing in vitro studies under aseptic conditions.
In vitro testing of pathogenicity: In vitro regenerated guava leaves were used to determine the phytotoxic effect of Fusarium sp. cell free culture-filtrate. Leaf segments (approximately 20 mm) of 30 day-old in vitro regenerated guava plants were taken and punctured with a fine needle and 5 μL concentrated Fusarium sp. phytotoxic culture filtrate (90% v/v) was dropped onto the wounded area. All leaf segments were incubated on moist what man filter paper No. 1 (analytical grade) in sterilized petri dishes under continuous fluorescent light at 26±2°C. Sterile water and Potato dextrose broth were used as controls and each test was repeated three times. Symptoms of leaves were noted after 72 h of incubation.
Callus growth bioassay: Callus cultures of guava were established using
immature zygotic embryos as initial explant for callusing from cultivar Allahabad
Safeda and placed on Murashige and Skoog medium (Murashige
and Skoog, 1962) basal medium supplemented with 2.0 mg L-1 2,
4-D, 400 mg L-1, 60 g L-1 sucrose and solidified using
8 g L-1 agar. Ten pieces of guava calli approx. Ten milligram were
transferred to induction medium. The semisolid medium was supplemented with
various concentrations of culture filtrate (0, 5, 25, 50 and 100%) and control
without culture filtrate containing medium. The experiment consisted of three
replicates for each experiment in a completely randomized design. Calluses were
incubated for up to four week and symptoms were noticed and ranked into five
categories: 1 = browning, 2 = friable callus, 3 = dried callus, 4 = deeply brown
with no regeneration, 5 = compact and regenerating.
Culture procedure: In vitro somatic embryogenesis derived plantlets of guava cv. Allahabad Safeda were inoculated on full strength Murashige and Skoog (Hi-media) liquid media (devoid of any growth hormone) containing various concentrations of culture filtrate (0, 5, 25, 50 and 100%, v/v) of Fusarium sp. and control without culture filtrate. Ten selected pure pathogenic cultures of Fusarium oxysporum f. sp. psidii and Fusarium solani were used as selection agent for screening guava plantlets for developing wilt resistance under in vitro conditions. Each experiment was conducted in a completely randomized design with five replications and each experiment carried 50 explants/treatment and repeated at least thrice for further statistical analysis (O.P. Sheoron-A software).
In vitro selection using callus culture and plantlets: Approximately
15-20 callus/treatment and at least 50 plantlets per treatment were taken and
subjected to recurrent selection in Murashige and Skoog liquid media (without
agar) culture with a modified culture medium containing 0, 25, 50 and 100% (v/v)
fungal (Fusarium sp.) filtrate/culture filtrate. The MS liquid medium
were prepared autoclaved and cooled at room temperature and the Fusarium
sp. culture filtrate were amended into the liquid media after cold-filter-sterilization
through 0.22 μ Millipore syringe filter and the pH of the culture filtrate
was adjust to 5.8±2 before adding to the sterile MS liquid medium in
order to eliminate loss of activity of culture filtrate during autoclaving.
The callus cultures and plantlets were subculture once at every 10th day and
during each subculture, fresh medium with culture filtrate/culture filtrate
were used. After four and five such transfers, surviving callus or plantlets
were recovered and cultured in medium without culture filtrate for further proliferation
and those survived were considered as putative resistant and subjected to subsequent
selection cycles. The cultures were incubated at 28±2°C in the dark
for four selection cycles (each selection cycle of 21 days).
In the current study, ten most virulent/pathogenic cultures filtrates of Fusarium sp. five of Fusarium oxysporum f. sp. psidii and five of Fusarium solani selected for carrying out in vitro selection process. Of these ten culture filtrates; three of the culture filtrates (F2, F15 and F20) of Fusarium solani were proved to be a potent selection agent (Fig. 1, 2) shows promising selections in both the explants viz., callus and regenerated plantlets. The percentage survival of callus cultured on media containing culture filtrate at various concentrations decreases with increasing concentration, reaching a 0% survival at 100% culture filtrate (Fig. 3).
In some Fusarium sp. culture filtrate F18 and F24 (Fusarium oxysporum
f. sp. psidii) a 100% plantlet regeneration or survival were noticed
with increasing concentration of culture filtrate even after four selection
cycles (Fig. 4d). Although, the shoot regeneration in treated
plantlets was observed after 2 weeks of treatment. However, in case of explant
callus regeneration frequency after two selection cycles were found varied significantly
with respect to the concentration of Fusarium sp. culture filtrate.
Lowest regeneration efficiency of callus was observed in 100% culture filtrate
of Fusarium sp. as compared to the control. The gradual selection of
the plantlets for resistance to higher concentration of culture filtrate were
apparent as the plantlets finally surviving on the culture filtrate at higher
concentration 50% of culture filtrate describes promising selections as well
as shows maximum wilting symptoms (Fig. 4a-d)
with higher concentrations resulted in a small proportion of viable shoots which
may be adopted to develop resistant genotypes.
||In vitro selection in guava using culture filtrate
of Fusarium solani (F2, F15 and F20) shows a series of wilting symptoms;
F2a: Mottling of leaves and root zone, F2b: Browning of root zone, F2c:
Drying of leaf at tip portion and ultimately death of plantlets, F15a: Mottling
of root-zone, F15b: Browning and blackening of root-zone, F15c: Drying of
upper leaves, F20a: Falling leaves, F20b: Leaves drying and falling of leaves
and F20c: Drying of root zone and yellowing of leaves
||Depicting in vitro selection system in guava using Fusarium
oxysporum f. spp. psidii at 50% culture filtrate (F18) shows
plant growth promoting activity and no wilting symptoms (Fig.
2a-c) formation of fibrous roots and growth of leaves
||Different stages of callus mortality symptoms when exposed
to 50% Fusarium solani (F2) culture filtrate amended in medium
shows maximum wilting, (a) browning of callus with loss of embryogenesis,
(b) callus blackening, (c) friable and watery callus, (d, e) dying of fragile
callus and (f) drying and death of callus tissue with no further regeneration
||In vitro screening of guava plantlets cv. Allahabad
Safeda (Psidium guajava L.) using culture filtrates of 10 isolates
of Fusarium spp., (a) Percent survival of plantlets at 5% culture
filtrate, (b) Percent survival of plantlets at 25% culture filtrate and
(c) Percent survival of plantlets at 50% culture filtrate during four selection
According to the present findings Fusarium solani culture filtrate F2,
F15 and F20 culture filtrate (Fig. 1) shows maximum wilting
symptoms with increasing concentration starting from 0, 25, 50 and 100% conversely,
in the controls. At control 100% plantlet regeneration was recorded and no symptoms
of mortality (Fig. 4a-d). The plantlets
selection were therefore carried out at the 50% culture filtrate level to exert
maximum selection pressure that can allowed the recovery of resistant plantlets.
Additionally, in the present study the other two culture filtrates of Fusarium
oxysporum f. sp. psidii (F18 and F24) poor symptoms of wilting even
after 30 days or fourth week of a selection cycle (Fig. 2).
However, these two selections (F18 and F24) showed good regeneration at maximum
(100%) concentration of culture filtrate after four weeks of selection (Fig.
4d). This assumes that Fusarium oxysporum f. sp. psidii isolates
used in this study was non-pathogenic or avirulent as compared to the Fusarium
solani which is registered as a most virulent pathogen causes serious damage
to guava plants which is observed from this study. Pathologically, it has been
proved that Fusarium solani is the most virulent and pathogenic in nature
found commonly wherever wilt disease is to be reported (Gupta
et al., 2010). The results of current study revealed that some 15-20%
of guava calli shows ceased growth or become friable and become severely blackened
and 80% calli was mortalized after first selection cycle (data not given). Figure
3a-f clearly demonstrates the callus mortality symptoms
as well and the remaining or survived cultures exposed to selection pressure
after first selection cycle shows poor regeneration or friability.
This study reports the first attempt to assess the potential of Fusarium
sp. culture filtrate as a potent selection agent for establishing in vitro
selection system in guava (Psidium guajava L.) cultivar Allahabad Safeda
for resistance to Fusarium wilt. The data demonstrate that this approach
could be potentially employed for screening and establishing large populations
of the guava cultivars for resistance to Fusarium wilt in Indian sub-continent.
Histopathological studies confined that no traces of Fusarium mycelium
was found from the wilted guava plant. Presumably, it was concluded that the
toxins released by the Fusarium sp. in the xylem vessels causes yellowing
of upper leaves, wilting of stems and eventually resulting in the death of a
plant (Beckman and Roberts, 1995). In vitro selection
approach using Fusarium sp. culture filtrate appears as a promising tool
to rectify specific defects to improve a desirable trait of highly adapted cultivars
without involving a sexual cycle. Chandra et al. (2010a)
reviewed the in vitro selection as candidate approach to select disease
resistant lines in culture using phytotoxic fungal culture filtrate or purified
toxins produced by the pathogen (Behnke, 1979; Wenzel,
1985; Daub, 1986; Chawla and
Wenzel, 1987; Jayasankar and Gray, 2003). The availability
of a defined culture filtrate or pathogen culture filtrate has been advantageous
for inculcating in vitro selection protocol. Culture filtrates that are
known to play a role in pathogenesis are called pathotoxins (Bulk,
1991; Svabova and Lebeda, 2005). However, to be
useful, toxins must be implicated in the disease development process which act
at the cellular level and have a mode of action that allows recovery of resistance
(Yoder, 1980; Daub, 1986; Hammerschlag,
1984; Svabova and Lebeda, 2005).
The correlation between guava genotype susceptibility and the toxicity of culture
filtrates suggests that filtrates could be used as potent selection agent to
screen in vitro regenerants for disease resistance. Tissue culture systems
can provide a means of rapid screening for disease resistance when a culture
filtrate is involved in disease development (Daub, 1986;
Hammerschlag, 1984; El-Hadrami et
al., 2005). However, it has been concluded from the present findings
that Fusarium sp. culture filtrate can have enough potential to be used
as a selection agent against wilt resistance in guava cv. Allahabad safeda.
Accordingly, Jin et al. (1996) obtained culture
filtrate from Fusarium solani which has pathogenic characteristic, inhibit
the growth of soybean cultures which was susceptible to Sudden Death Syndrome
(SDS) disease. Similarly, in case of apple (Raman and Goodwin,
2000), mango (Jayasankar et al., 1999), peach
(Hammerschlag, 1988), strawberry (Toyoda
et al., 1991), lemon (Gentile et al., 1992)
and pineapple (Borras et al., 2001), banana (Matsumoto
et al., 1999; Okole, 1995), grapevine (Jayasankar
et al., 2000) in vitro selection system has been carried
out successfully to develop a resistant lines and varieties. An important biotechnological
aspect of crop improvement via an in vitro selection approach is that
traits selected at the cellular and plant level must be expressed in the regenerated
plants. Our results showed that guava plantlets selected using Fusarium
sp. culture filtrate against Fusarium wilt at various concentrations
on callus and in vitro regenerated plants exhibited improved resistance.
It was also concluded from previous studies that A number of previous studies
that toxins present in the culture filtrate were able to inhibit growth and
survival of the plantlets of the host species were more sensitive to the toxin
than those of non-host plants (Huang and Hartman, 1998).
According to our findings, about 15% of the plantlets exhibited resistance to
Fusarium solani and the plantlets having resistance were further shifted
to green house conditions for hardening to be developed as resistant/tolerant
plantlets. In both the susceptible and resistant plantlets, increasing the concentrations
of culture filtrate increased the intensity of response which was measured by
symptoms of wilt i.e., burning of upper leaves, leaf fall, mottling of leaf,
browning of root-zone.
Hence, the present investigation illustrates the development of a recurrent selection system in vitro which can possibly an alternative solve towards the long withstanding problems in guava like susceptibility to Fusarium wilt of guava and which can be successfully addressed especially in popular and commercial variety like Allahabad Safeda which has desirable traits in all other aspects except resistance to Fusarium wilt disease. A stable in vitro selection system developed on the basis of the current findings can go a long way in eliminating single trait problem while retaining others and also, shortening the time to release a variety which otherwise would entail a long period of 10-12 years by conventional means.
Authors are highly thankful to Director, C.I.S.H., Rehmankhera Lucknow, India for providing necessary research facilities and preparation of manuscript is gratefully acknowledge. Author (Madhu Kamle) is highly thankful to Dr. A. K. Misra, All India Project Co-coordinator for Subtropical Fruit Crops, C.I.S.H., Lucknow for providing the Fusarium sp. cultures for carrying out the present study which is a part of her Ph.D thesis.
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