Physiological changes associated with Okra mosaic virus infection in field grown okra plants

As a prerequisite for the breeding of virus-resistant okra cultivars, the effect of Okra mosaic virus infection on physiological performance of ten okra cultivars were assessed under field conditions within the coastal savannah agro-ecological zone of Ghana. All cultivars succumbed to the disease and expressed varying degrees of symptoms. The results showed that plant height at initial and 50% flowering were significantly reduced with cultivar Clemson spineless showing the highest reduction of 38% and 35% respectively. Plant height at 50% podding was also reduced in all cultivars with the highest reduction (40%) occurring in cultivar Clemson spineless. Virus-infected plants of cultivars Kwabenya 1, Labadi dwarf and Asutem took on the average 43 days to attain 50% flowering compared to 25 days in healthy or control plants. Diseased plants had significantly (P<0.05) delayed, taking on the average 47 days after planting to attain 50% podding compared to the control plants with an average of 35 days. Infected plants of cultivars Kwabenya 1 and Labadi dwarf took the highest number of 51 days to attain 50% podding. Pod length and width were also reduced in all cultivars but were highest in cultivars Lucky 19 F1 and Clemson spineless at 52.19% and 46.25% respectively. Based on this assessment, cultivars Adom and Asutem which were less affected in terms of disease severity and fruit yield reduction could be exploited in future breeding programmes.


Introduction
Okra (Abelmoschus esculentus L. Moench) is grown for its immature fruits and fresh tender leaves.These plant parts serve as food and an important source of raw materials for some pharmaceutical and other industrial applications due to the high phytochemical and nutritional content (Adetuyi et al., 2011).Okra is a robust crop, hence is able to survive harsh conditions where most other crops fail (Oppong-Sekyere et al, 2012).This unique agronomic quality coupled with its rich nutritional qualities make it a very popular crop for resource-poor small-holder farmers in rural areas across tropical Africa.
Notwithstanding the importance of okra as a domestic and export crop, its production is constrained by severe crop damage and low yields due to viral diseases (Chen et al., 2019;Asare-Bediako et al., 2014;Savary et al., 2012), hence the full food, medicinal and economic potentials of the crop are not realized.The most important viruses of okra include Okra Yellow Vein Mosaic Virus (OYVMV), Okra Mosaic Virus (OkMV) and Okra Leaf Curl Virus (OLCV).These viruses are widespread in tropical and subtropical regions across the world.OkMV has been reported as widespread in Ghana, resulting in significant yield losses (Appiah et al., 2020).The virus is a member of Tymovirus group, characterised by a single stranded DNA (ssDNA) gonome and spread by the flea beetle Podagrica spp (Asare-Bediako et al., 2014).These important okra viruses infect okra plants either singly or mixed.According to Hull (2002), mixed viral infections could be either antagonistic or synergistic.Antagonism normally arises when co-infecting viruses are related resulting in cross protection (Aguero et al., 2018) or interference (Kumar et al., 2016).Synergism also arises when co-infecting viruses are not related, resulting in more severe disease symptoms than those caused by single infections (Cho et al., 2000).Studies done by Ndunguru and Rajabu (2004), revealed that okra exhibits a variety of symptoms which usually ranged from mild to severe, caused by either single or mixed virus infections.In mild situations, okra show symptoms such as veinal mottling, vein banding and leaf mosaic.In severe cases, however, infected plants show small and twisted leaves, downward curling of mature leaves, deformed fruits and stunted growth.A large variety of okra cultivars exist in Ghana and are predominantly cultivated across the major okragrowing regions of the country (Ahiakpa et al., 2017).These cultivars include exotic types sold by commercial seed companies with several trade names.Unfortunately, almost all known cultivars of okra currently cultivated in Ghana, both local and exotic are highly susceptible to the major viruses and many species of insect pests (Oppong-Sekyere et al, 2012;Prakasha et al., 2010;Petlamul et al., 2009).The implication of this is that production levels of okra have been generally very low.Although, some works have already been done in Ghana, most of these studies have focused on viral disease epidemiology, little is known about the effects of viral diseases on growth and performance of different okra cultivars.There is, therefore, the need to evaluate the effects of OkMV on cultivated okra cultivars present in Ghana in order to be able to recommend the best cultivars for farmers in the short term and in the long term, those to be incorporated into breeding programmes to expand the genetic variation of the crop.The present study, therefore, sought to evaluate the effects of OkMV infection on ten okra cultivars in order to identify better performing cultivars.

The study area
The research was conducted on the research fields of the Biotechnology and Nuclear Agriculture Research Institute (BNARI) of the Ghana Atomic Energy Commission (GAEC).The study area is located at Kwabenya, Accra on latitude 5º 40' N, longitude 0º 13' W with Ochrosol (Ferric Acrisol) soil type, derived from quartzite Schist.The site is well drained and has an elevation of 76 m above sea level within the coastal savannah agro-ecological zone (Dickson and Benneh, 2004).The maximum and minimum average temperatures for the period of study were 30.7 and 26.0 o C respectively with average annual rainfall of 220 mm.The highest and lowest relative humidity were 75 and 60% respectively (Dickson and Benneh, 2004).

Planting materials and field design
Planting materials used comprised five exotic okra cultivars namely; Lucky 19F1 (LF1), F1 Kirene (FIK), F1 Sahari (F1S), Kirikou F1 (KF1) and Clemson Spineless (CS) obtained from Technisem, Accra and five local okra cultivars comprising Asutem (AS), Togo (TG), Labadi dwarf (LD), Kwabenya 1 (K1) and Adom (AD) obtained from the local markets and okra farmers' fields.After land preparation and field demarcation, the okra seeds were directly sown to a depth of 2 cm at a spacing of 0.50 m x 0.60 m between and within rows in a Randomized Complete Block Design (RCBD) with four replications.Each replicate measured 35 m x 7 m and were separated by 2 m from each other.Each block consisted of 10 subplots with each subplot measuring 3 m x 3 m and spaced 1 m from one another.Four seeds per hill were sown and later thinned to one seedling after emergence.Cultural management practices such as weed control and watering were done as and when required.

Data collection
Ten plants were randomly selected from the middle rows of each plot per cultivar and tagged.The ten selected plants consisted of five healthy or control plants and five infected plants.Symptom severity was scored on the diseased plants following a five-point scale (0 to 4) where; 0 = No symptom; 1 = Very mild symptoms, initial vein clearing, initial leaf yellowing, mild curling and blistering; 2 = Leaf completely yellow and inter-veinal regions remain green or yellow and blistering; 3 = Severe curling, yellowing, stunting and blistering; and 4 = Severe yellowing, curling, blistering and deformed pods (All leaves of the plants affected).Data was collected on quantitative traits with respect to reproductive and pod characters.The reproductive characters comprised Days to 50% flowering, Days to 50% podding, Plant height at initial flowering stage, Plant height at 50% flowering stage, and Plant height at 50% podding stage.Days to 50% flowering and Days to 50% podding were determined by counting the days after plant emergence.On the other hand, plant height at initial flowering stage, plant height at 50% flowering stage, and plant height at 50% podding stage were measured using tape measure and recorded in centimetres (cm).Chlorophyll contents of the cultivars were measured using hand-held SPAD meter.The pod characters that were recorded included pod length, pod width and fresh fruit weight.The pod length and width were taken with a tape measure and the mean for five plants recorded.Fresh fruit weight was measured with a spring balance and the mean recorded in kilogram (kg).

Statistical analysis
Genstat Statistical Software Package (12 th edition), and Microsoft Excel Software (2010 edition) were used for the data analyses and a p-value of 0.05 or less was considered as statistically significant.The data collected on diseased and healthy plants were subjected to Analysis of variance (ANOVA) and the Tukey's pairwise comparison test was used to determine the differences among the means.

Viral disease symptom severity
Disease symptoms were found mostly in the young leaves and included leaf yellowing, leaf malformation, vein clearing, mosaic, leaf blistering, upward cupping of leaves and stunting (Fig. 1).Generally, symptom severity increased steadily from week 3 to week 13 for all cultivars (Fig. 2).Cultivar Kwabenya 1 and Lucky19 F1 expressed the most severe symptoms of 3.85 and 3.61 respectively at week 13, although this was not significantly different from the others.The least symptom severity score (2.0) at week was found in cultivar Adom and Asutem which recorded mild symptoms throughout the study period.

Influence of OkMV on plant height at initial flowering stage
OkMV infection influenced plant height at initial flowering stage.Generally, plant height was significantly (P<0.05)reduced in diseased plants of all cultivars.The reduction in plant height was most severe in cultivars CS (38%), F1K (35%), AD (35%) and F1S (24%) (Fig. 3).The overall percentage reduction in plant height of diseased plants compared to healthy plants was 19.7%.

Plant height at 50% flowering stage was significantly as affected by OkMV infection
Plant height at 50% flowering was significantly (P<0.05)influenced by the effects of infection of OkMV (Fig. 4).In all cultivars, at 50% flowering, plant height was significantly (P<0.05)reduced in diseased plants.The reduction in plant height was more pronounced in cultivars AD (24%), CS (35%), F1K (28%) and F1S (26%) whilst KF1 had the lowest percentage reduction in plant height (13%).The overall percentage reduction in plant height of diseased plants compared to the healthy ones was 20.4%.

Plant height at 50% podding stage was significantly as influenced by infection of OkMV
The effect of OkMV infection on plant height showed significant (P<0.05)differences when healthy and diseased plants were compared at 50% podding (Fig. 5).Plant height reduction was more pronounced in the cultivar CS (40%) and least in KF1 (20%).The overall percentage decrease in height of diseased plants was 14.9%.

Days to 50% flowering as influenced by OkMV infection
Generally, diseased plants took significantly longer time to start flowering irrespective of the cultivar (Fig. 6).Diseased plants of cultivar K1, LD and AS took averagely the highest number of days (43 days) to attain 50% flowering.A second group comprising F1K, TG, F1S, CS, AD, KF1 and LF1 attained 50% flowering earlier at an average number of 38 days.On the other hand, healthy plants of cultivars TG, F1S, F1K and AS took significantly the least number of days (25 days) to 50% flowering compared to the diseased ones.

Days to 50% podding as influenced by by OkMV Infection
Days to 50% podding was delayed by the viral infection in all the okra cultivars.Infected plants had significantly (P<0.05)delayed (average 47 days after planting) number of days to 50% podding compared to healthy plants with an average 35 days (Fig. 7).Infected plants of cultivars K1 and LD took the highest number of days of at least 51 days to attain 50% podding while cultivar F1K attained 50% podding significantly earlier at 35 days.Healthy plants of F1K, AS, LF1 and TG attained 50% podding earlier at average of 30 days after planting.However, when infected, attainment of 50% podding was delayed for about 16 days except F1K which attained 50% podding at 36 days even when diseased.

Pod length as affected by OkMV infection
In all cultivars, virus infection caused significant (P<0.05)reduction in pod length.However, the effect of the disease on pod length reduction was much pronounced in TG where the average pod length of healthy plants was 50% longer than pods of diseased plants.Similarly, in cultivars LF1, K1 and KF1, average pod length decrease of 45%, 30% and 25% respectively were estimated.Diseased plants of cultivar AS produced significantly, the shortest pod lengths.
With respect to percentage reduction in pod length, accession LF1 had the highest (52.19%) whereas F1S had the lowest (26.44%) (Table1).Each value is a mean of four replicates, and values followed by the same superscript(s) are not significantly different (p>0.05) according to Tukey's pairwise comparison.

Pod width as influenced by OkMV infection
The effect of OkMV infection on pod width reduction was significant (P<0.05) (  produced significantly the highest pod widths of 9.65±1.17and 8.80±0.90respectively compared to diseased plants of other cultivars.

Fresh pod weight as affected by OkMV infection
Fresh fruit weight was significantly (P<0.05)reduced by the effect of OkMV (Table 3).Generally, viral infection caused an estimated 50% reduction in the fresh fruit weights comparing healthy and diseased plants.Cultivar F1K was found to produce the heaviest fresh fruit weights while diseased plants of F1S, LD and AS produced significantly the lightest fresh fruit weights.Cultivars that produced intermediate fresh fruit weights from diseased plants include AD, CS, K1, KF1, LF1 and TG.Highest percentage reduction in fresh fruit weight was seen in cultivar LF1 (52.19%) whilst LD (28.45%) had the lowest.

Correlation among four essential traits in ten okra cultivars
Correlation coefficients were worked out to study the relationship of chlorophyll content (CC) with fresh fruit weight (FFW), pod width (PW) and pod length (PL).
The results of correlation coefficients indicated that chlorophyll content shared very strong positive correlation with fresh fruit weight (FFW) and pod width (PW) but weak negative correlation with pod length.Fresh fruit weight (FFW) exhibited very weak correlation with pod length (PL) but very strong positive correlation with pod width (PW) while Pod length (PL) and pod width (PW) shared very strong positive correlation.

Discussion
It is noteworthy that the ten okra cultivars assessed in this study were previously evaluated for Okra mosaic disease resistance in which all cultivars succumbed to the OkMV infection (Appiah et al., 2020).In the current study, symptom expression among the cultivars was severe, perhaps due to cultivar susceptibility, strain of the virus and probably the influence of other viruses which were not detected in this study.The infection resulted in different kinds of symptoms which culminated in poor plant growth.The reduction in plant growth and yield as a result of virus infection has been reported (Chen et al., 2019;Latham et al., 2004).Generally, plant height at initial flowering stage, 50% flowering and 50% podding stages was significantly reduced in diseased plants of all cultivars.The average percentage reduction in plant height of diseased plants compared to healthy plants at initial flowering stage was 19.7%.This result corroborates the findings of Ndunguru and Rajabu (2004) who recorded 19.5% reduction in plant height between virus-infected and healthy plants.In most of the cases, stunting was severe, probably due to mixed infections with other viruses.Cho et al. (2000), observed severe plant damage as a result of infection caused by two or more viruses.However, it must be noted that the disparity in the percentage reduction in plant height observed in the different cultivars in this study could be a result of the differences in genetic make-up of the cultivars resulting in differential reactions to the virus.Reduction in the height of virus-infected plants have been reported in groundnuts (Appiah et al., 2016), cowpea (Kareem andTaiwo, 2007) and pepper (Pazarlar et al., 2013).Generally, flowering was delayed in diseased plants of all cultivars compared to healthy or control plants.Diseased plants took significantly longer time to flower irrespective of cultivar.Delay in flowering in tomato plants infected with Potato virus X and Tobacco mosaic virus (Balogun, 2003) and okra plants infected with OkMV (Ndunguru and Rajabu 2004) has been reported (Ndunguru and Rajabu, 2004).The delayed flowering observed in diseased cultivars in this study could be due to impaired photosynthesis, ineffective uptake of nutrients, and other stress conditions imposed by the disease during the growing period of the cultivars (Niser et al., 2002).As in days to 50% flowring podding was significantly affected by the viral infection in all the okra cultivars.The infection significantly delayed the number of days to 50% podding compared to healthy plants by an average of 35 days.Viruses have a remarkable impact on their host metabolism, with carbohydrate and chlorophyll metabolism being two activities which are seriously affected by viral infections.Chlorophyll content decreased in virus-infected plants of all the cultivars.Lower levels of chlorophyll observed in the virusinfected plants in this study may be due to inefficient functioning of chlorophyllase, leading to poor development of chloroplast or inhibition of chlorophyll synthesis as reported by Zhao et al., 2016 andRoca andMinguez-Mosquera (2003).Furthermore, the chlorotic and mosaic symptoms observed on the virus-infected plants could indicate the occurrence of chloroplastvirus interactions (Zhao et al., 2016;Rahoutei et al., 2001), resulting in fluctuation of chlorophyll fluorescence and reduced chlorophyll pigmentation (Liu et al., 2014).The reduction in the chlorophyll content may have resulted in reduced photosynthetic activity culminating in low yields.Parimala et al. (2009) reported a decrease in yield of virus-infected okra as a result of reduced chlorophyll content.In this study, chlorophyll content showed a strong positive correlation with fresh fruit weight.There has been a growing interest on the part of plant breeders to develop higher yielding varieties of crops (Nestel et al., 2006;Gregorio, 2002).Therefore, information on the association between the chlorophyll content and yield Samuel Amiteye et al 8/10 Asian J Agric & Biol.2021(4).
parameters will serve as a useful guide in future breeding efforts.Results from the correlation analysis implies that targeting high chlorophyll content of okra leaves through breeding will consequently lead to improvement in fresh fruit weight and pod width.Significant reduction in pod length, width and weight was observed in virus-infected plants of all the cultivars.Generally, for all cultivars; local or exotic, healthy or control plants produced pods with bigger widths than pods produced from diseased plants.It is noteworthy that despite the infection, diseased plants of cultivar AD and K1 produced significantly higher pod widths than diseased plants of other cultivars.This may suggest that these cultivars are tolerant to viral infections and therefore may offer some bases for their selection for introgression of desirable genes for high fruit yield into preferred cultivars.Tolerance of okra cultivars to OYVMV have been reported (Mohsan et al., 2017).OkMV infection reduced fresh fruit weight of all cultivars.The viral infection resulted in an estimated 50% reduction in the fresh fruit weights compared to weights recorded in healthy plants.The reduction in the fruit weight of the virus-infected okra cultivars observed in this study could be due to reduction in photosynthesis (Liu et al., 2014) resulting from the viral infection.Differences in the average fresh pod weight per plant observed among the cultivars may be attributed to inherent fruit characteristics such as higher pod diameter, higher number of pods per plant, fruit length, and higher number of branches per plant (Mahapatra et al., 2007;Mohammad et al, 2001 andSingh andJain, 2002).Pod length, pod width and fresh fruit weight are desirable characteristics for domestic, industrial and commercial preference (Alam and Hossain, 2008).Results from the current study indicate that pod length, pod width and fresh fruit weight were considerably decreased in diseased plants compared to records of these characters in the healthy or control plants.Furthermore, reports by Echezona and Offordile (2011) attributed the cause to the feeding activity of Podagrica spp. that causes damage to the leaves resulting in significant reduction in the photosynthetic capacity.Similar reduction in the yield of okra cultivars as a result of OkMV infection has been reported (Fajinmi and Fajinmi, 2010).In addition to the effect of OkMV on growth and performance of the okra cultivars, the result of this study has also revealed significant variation among cultivars with respect to pod length, pod width and fresh fruit weight.This implies a broad range of genetic variability which could be exploited in breeding programmes.Singh et al. (2017), observed significant variation among okra cultivars, suggesting variations that are useful for studying genetic diversity of okra germplasm.Cultivars K1, AD and F1K are the best with respect to pod length, pod width and fresh fruit weight and therefore can be selected as parents in future breeding work.

Conclusion
This study has elucidated the effect of okra mosaic disease on the growth and performance of ten okra cultivars under field conditions within the coastal savannah agro-ecological zone of Ghana.All ten okra cultivars succumbed to the disease but had varying levels of reduction in growth and performance.Diseased plants compared to healthy ones had significantly reduced (p≤0.05)chlorophyll content, plant height, delayed flowering process and fresh pod weight.Lower levels of chlorophyll content were observed in the virus-infected plants with consequent reduction in photosynthetic activity culminating in lower yields.Plant height of diseased plants was significantly reduced compared to healthy or control plants at initial flowering stage.The viral infection also significantly delayed the achievement of 50% podding by an average of 35 days.Similarly, fresh fruit weights of diseased plants decreased by an estimated 50% compared to weights recorded in healthy plants.The observed significant reduction in the yield parameters caused by OkMV disease emphasises the damaging potential of the disease on okra cultivation and the need for improved control measures to maximise production.Two local cultivars, Adom and Asutem were found to be the best performing cultivars under field conditions having recorded less reduction in fresh fruit weight despite the infection.Therefore, these cultivars can be selected as locally-adapted, good materials tolerant to OkMV for further improvement in future breeding work.

Figure- 1 .
Figure-1.Viral disease symptoms found on the okra cultivars A: Healthy plant, B: mosaic, blistering and upward cupping of leaves, C: vein clearing, D: leaf malformation, E: mosaic and F: leaf yellowing, upward cupping of leaves and stunting.

Figure- 3 .
Figure-3.Effect of OkMV infection on plant height at initial flowering stage.Bars represent the standard error of means of four replications.

Figure- 4 .
Figure-4.Effect of infection of OkMV on plant height at 50% flowering stage.Bars represent the standard error of means of four replications.

Figure- 5 .
Figure-5.Effect of infection of OkMV on plant height at 50% podding stage.Bars represent the standard error of means of four replications.

Figure- 6 .
Figure-6.Effects of infection of OkMV on days to 50% flowering.Bars represent the standard error of means of four replications.

Figure- 7 .
Figure-7.Effects of infection of OkMV on days to 50% podding.Bars represent standard error of four replications

Figure
Figure-8.Effects of OkMV infection on the chlorophyll content of diseased plants of cultivar Clemson spineless.

Table 2
).Generally, all cultivars, local and exotic, healthy plants produced pods with bigger widths than pods produced from diseased plants.Healthy plants from F1K and AD produced significantly the biggest pod widths.On the other hand, diseased plants from TG, AS and CS cultivars produced the least pod widths.Percentage reduction in pod length was observed to be most pronounced in KF1 (35.22%) and least in AD (26.89) (Table2).Diseased plants of cultivar AD and K1