Certain growth related attributes of bunchy top virus infected banana under ex-vitro conditions

Effect of banana bunchy top virus (BBTV) on morpho-physiological characteristics of banana (Musa sp.) cv., Basrai plants was assessed. Healthy and BBTV infected samples of banana were collected from its open fields and micro-propagated aseptically. These plantlets were established in wire-house for three months. Presence of BBTV was confirmed by polymerase chain reaction (PCR) and enzymelinked immunosorbent assay (ELISA). Micro-propagation efficiency of BBTV-infected plants was observed in less than healthy plants under in-vitro as well as in-vivo. Significant reduction in plant height, fresh weight (F wt) and dry weight (D wt) of leaf was observed in BBTV infected plants. A corelation of peroxidase (POX) activity was observed with total carotenoids that increased in BBTV infected plants, while chlorophyll contents decreased significantly. Nitrate reductase activity also decreased with increase in proline contents in BBTV-stresses plants (p<0.05). Meanwhile, reducing sugars also increased but not-significantly. Bunchy top virus infection in banana therefore resulted in alteration of growth related physiological traits that led to retardation of plant growth.


INTRODUCTION
In plants, a number of environmental stresses (biotic and abiotic) produce characteristically identifiable symptoms because of deleterious impacts on different physiological processes.Each stress has developed a number of biochemical changes (Miteva et al., 2005).Viral pathogens (biotic stress) have also caused severe damage in many major crops either by reducing yields or quality of plants as a whole (Kang et al., 2005).Presently, banana is affected by five growth limiting viruses (Stover, 1972), with the most destructive being the banana bunchy top virus (BBTV) transmitted through planting infected banana nursery and by banana aphids (Pentalonia nigronervosa) to healthy plants.This virus is observed around the world, especially in Asia and the Pacific regions (Magee, 1940;Wu, 1978;Dale, 1987;Su, 1990;Harding et al., 1991;Moffat, 2001).Symptoms of this viral infection become visible when virus severity in infected tissues reaches to critical point.The BBTV infected plants have been noted with stunted growth, deformation of young leaves and bunched at top of plant (Chia et al., 1992(Chia et al., , 1995;;Hendry, 1987).Virus-free plants show relatively better vegetative growth in field, but reduce with viral infection due to decreased photosynthetic surface, as well as respiration rate (Balachandran et al., 1997;Guo et al., 2005).In general, the plant growth of virus infected plants is limited because of inhibitions of biological system in communication to growth related metabolic processes.Similarly, POX enzymes play an integral role in plant metabolism and immune system from seed germination to maturity and become highly active during senescence (Siegel, 1993).The plants immune system is primarily dependent on POX and show hypersensitivity response.Systemic viral infection may lead to increase in defense activity, as well as wound repair by POX (Ye et al., 1990;Candela et al., 1994).Meanwhile, necrosis or chlorosis appears by virus infection (Wood, 1990).In higher plants, both biotic and abiotic stresses have produced a number of characteristic changes in plant morphs and metabolic processes.For example, tissues' POX activity increases because of toxic elements present in cells (Espelie et al., 1986;Stroinski, 1995;Miteva et al., 2005).
In the present experiment, morpho-metabolic attributes were quantified in BBTV infected and virus-free banana plants (Musa sp.) comparatively.BBTV infection developed a number of specific abnormal traits in multiplied banana plants under in-vitro and ex-vitro also.Identification of these characters may be helpful to the studies of future amelioration for BBTV resistance in banana crop.

MATERIALS AND METHODS
The BBTV infected and healthy suckers of banana (Musa sp.) cv., Basrai were collected from different open field banana farms.Inner meristematic regions were excised from suckers and used as explants for micro-propagation (Haq and Dahot, 2007a, b).Micropropagated plantlets were propagated for three sub-cultures and rooted.These plantlets were screened for presence of BBTV through enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) (Figure 2) (Ennos, 2000;Haq et al., 2009) and shifted to wire-house for further development.When plants were of three months old, they were subjected to different morpho-physiological studies (Figure 1).The location of this experiment was far from the nearest banana field about 60 km.Exvitro propagating plants were sprayed twice (one-month interval) with imidacloprid (Provado ® ) at 60 ppm to control banana aphids by using small farmer's hand-pump (Robson and Wright, 2007).
Number of leaves was counted and plant height was taken from the sucker to petiole of fully emerged uppermost leaf and pseudo- stem diameter at centered point of plant height (Smith et al., 2000).Petiole related parameters were quantified from base of leaf blade to its attachment-point with pseudostem (Ennos, 2000).Leaf area (3 rd leaf from top) was measured by multiplying, maximum width of leaf with length and conversion factor (0.83).Fresh weight (F Wt) and dry weight (D Wt) of 100 leaf disks (6.3 mm) were taken and relative water contents (RWC) also calculated (Robinson and Neil, 1985;Conroy et al., 1988).Moreover, the third leaf from top of developing banana plants was used for different bio-chemical and anatomical studies.It was chopped into small fragments (2 to 3 mm in length) and fixed in fixation buffer [3.5% glutaraldehyde, 0.1 M phosphate buffer (pH 7.0)] for 10 h at room temperature.Mixture was washed with 0.1 M phosphate buffer twice and again fixed in osmium tetra-oxide (2.5%) overnight (Gielwanowska et al., 2005).Free hand sections were cut from the material for further analysis (Johansen, 1940).
In addition, chlorophyll contents were determined by using Arnon's method (1949), while for determination of POX activity, 1.0 g plant material was homogenized with 3.0 ml sodium phosphate buffer (66 mM; pH 6.1) according to the method of Ranieri et al. (1995) and analyzed as by Curtis (1971) and Bergmeyer et al. (1974).The nitrate reductase activity (NRA) was determined as 100 mg young leaf tissue in phosphate buffer.A reaction mixture was raised by mixing 1.0 ml of 0.1 mM potassium phosphate, 0.5 ml of 0.05 M potassium nitrate (KNO 3 ) and 1% isopropanol (v/v) with pH 7.5 at 30°C in dark.After 1 h, 1.0 ml sulfanilamide (1%) and 1.0 ml naphthyl ethylenediamine dihydrochloride (0.02%) were also added.The nitrate contents were quantified at 540 nm (Klepper et al., 1971).

Statistical analysis
Collected data was computed for significant measurements using COSTAT computer package (CoHort Software, Berkeley, USA) at 5% level.Each of the two types of banana plants, healthy (control) and BBTV (infected), had at least seven replicates.

RESULTS AND DISCUSSION
During this experiment it was observed that BBTV had differential effects on plant growth of banana from early stages to its maturity (Figure 1).Severity of symptoms increased with the increase in age of plant due to the decreased growth potential of developing plants under viral stress.Of course, leaf morphology was affected significantly.Petiole distance decreased (p<0.05), while its width and length also decreased but non-significantly.With the decrease in canopy size (non-significantly), leaf production rate was also low in the BBTV infected plants.Meanwhile, size of the structural cells also decreased (non-significantly), while leaf area decreased in BBTV infected plants significantly (Figure 3 and Table 1).Collectively, all of these characters resulted in the decrease in plant height of the BBTV infected plants (Ennos et al., 2000;Kang et al., 2006).
Imbalance ionic toxicity causes abnormalities in combination with all metabolic functions of plants.A   decreased order of K + , Ca 2+ and nitrate (NO 3 -) was observed to be interlinked with Na + and Cl -inverse proportionally (Table 2).Decrease in relative water contents (RWC) was also observed (Table 2).Deficiency in water contents causes a decrease in leaf turgor or hydraulic pressure on which various physiological processes and morphological traits depend like as stomatal opening and growth of leaves (Tecsi et al., 1996;Balachandran et al., 1997).Furthermore, the BBTV infection in banana caused a decrease in green pigments such as chlorophyll (Chl) a, b and ab.Systemic chlorosis due to BBTV infection causes a decrease in rate of photosynthetic processes in leaves.The Chl a have been considered as more sensitive than Chl b in BBTV infected plants (Table 1).The systemic infection caused by plant viruses may be acting as inhibitors for certain enzymes involved in biosynthesis of chlorophyll contents (Goodwin and Britton, 1988;Tecsi et al., 1996).BBTV continuously spread within vascular system and reduces plant growth or accelerate senescence.Moreover, total carotenoid contents increased significantly, which was a typical sign of senescence (Sutic and Sinclair, 1990;Valjakka et al., 1999).
In addition, accumulation of reducing sugars in infected plants was higher than healthy plants as had been widely assumed to be a response of pathogen stress (Table 2).Similarly, significant increase in proline contents in the BBTV infected plants is also a well known indicator of environmental stress (Table 2).Increased proline content may ameliorate the impact of certain stresses (Dorffling et al., 1990;Csonka and Hanson, 1991).Generally, it has been considered that accumulation of proline content is a typical plant osmotic stress response marker, as well as against biotic stress (Table 2).Increase in free proline and protein contents in leaves plant have been observed to be activated naturally under specific hypersensitive response against microbes, including viruses (Goodman et al., 1986;Shalitin and Wolf, 2000).A relationship between reducing sugars, proline contents and total carotenoids was observed as each was increased in infected plants than healthy plants.Phenolic contents in infected plants were also higher than control plants (Table 2).
Moreover, plants are able to tolerate certain environmental stresses to an extent because of accumulation of either inorganic or low molecular weight organic compounds.Their relative contribution varies from plant to plant, but they play a crucial rule in the plants growing under various stresses (Ashraf and Harris, 2004).For example, POX in healthy plants were observed to remain inactive (covalent-bonding forms), but active (soluble and ionic-bonding forms) in BBTV-infected plants (Table 2).The POX activity was higher in infected plants, while nitrate reductase activity was low in BBTV infected plants.POX therefore remained active and developed a relationship with the chlorophyll contents.It is observed that when chlorophyll content decreases, POX activity increases in virus infected plants but proportioned to total carotenoids (Tables 1 and 2).These alterations are involved in developing pathogen resistance mechanisms (Kuroda et al., 1990;Wood, 1990;Chittoor et al., 1999;Milavec et al., 2001;Wang et al., 2010).
According to Kuroda et al. (1990), POX is responsible for chlorophyll degradation during senescence.Similarly, Smart (1994) reported that chlorophyll degradation occurs due to increase in hydrogen peroxide (H 2 O 2 ) and phenolic contents in leaves (Table 2).The horseradish POX is responsible for the catalyses of magnesium ion (Mg 2+ ) removal from the precursor of chlorophyll a and chlorophyllin (Azuma et al., 1999).Still, the breakdown of chlorophyll contents through POX pathways in the senescent leaves is dubious (Matile and Hörtensteiner, 1999).In senescent leaves, a variant POX activity has been observed in different plant species during plant growth.Johnson-Flanagan and Spencer (1996) also reported that at maturity stage, plant de-greening occurs.At that time the degradation of the green pigments was associated with increased activity of POX, while low chlorophyllase activity in case of canola has been measured.However, POX and an oxidase in thylakoids of barley leaf are activated for degradation of chlorophyll contents during senescence (Kuroda et al., 1990).

Conclusion
In this experiment, plant growth decreased in BBTV infected plants due to reduction in synthesis of photosynthetic pigments.Rate of photosynthesis was associated directly or indirectly with a series of metabolic activities.Systematic viral infection caused inhibition of photosynthetic related processes.Meanwhile, significant increase in total carotenoids occurred with the increase in severity of BBTV infection.Accumulation of carotenoids and activation of POX are dependent on the inhibition of different physiological processes due to viral infection.Such loss of biological systems therefore triggers the visibility of specific coloring and become first symptom of BBTV infection than loss of yield and death of plants.

Figure 1 .
Figure 1.In-vitro and ex-vitro multiplying BBTV-infected and healthy banana (Musa sp.) cv., Basrai plants under experimental conditions.a and b, 1 st micropropagation culture of the non-infected and BBTV infected suckers; c and d, 3 rd sub-cultures respectively; e and f, six weeks old healthy and infected banana plants growing in wire-house, respectively; g and h, 3 rd leaf from top to bottom of three-months old healthy and infected plants, respectively.BBTV, Banana bunchy top virus.

Figure 3 .
Figure 3. Different structural cells, ep, epidermal; mes, mesophyll; ae, aerenchyma; from three-months old BBTV infected plants of banana (Musa sp.) cv., Basrai.a and b, The structural cells of healthy and infected banana leaves; c and d, mesophyll cells of healthy and infected banana leaves respectively.