Effect of Leaf Age on Rice Yellow Mottle Virus Severity and Chlorophyll Content with Mechanical Inoculation and Vector Transmission Method

MATERIALS AND METHODS

Study site: The study was carried out in the screenhouse at Africa Rice Centre (Nigeria Station) Ibadan, between April 2008 and June 2009.

Isolate used: The RYMV isolate categorized as pathotype one, recommended for vector-rice screening experiment (Sere et al., 2008; Nwilene et al., 2009) was used in the study. This isolate was obtained from infected plants at the Plant Pathology Unit of Africa Rice Center, Cotonou, Republic of Benin.

Insect vectors used: Life adults of Locris rubra and Oxya hyla used for the trial were collected with a sweep net from Africa Rice Center fields at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. The insects were caged in a screenhouse to feed on cowpea plants for three weeks in order to lose infectivity.

Rice cultivar evaluated: The rice cultivar evaluated in the trial was Moroberekan, a moderately susceptible cultivar to RYMV. This cultivar was obtained from AfricaRice Plant Pathology Unit, Cotonou.

Isolate propagation: The isolate was first propagated on BG 90-2 (a highly susceptible cultivar), following mechanical inoculation of 21-day-old rice seedlings in the screenhouse. Four weeks after inoculation, leaves showing typical yellow mottle symptoms characteristic of RYMV infection were harvested and used to prepare the viral inoculum. The inoculum was prepared by grinding infected leaf samples in 0.01 M phosphate buffer (pH 7) in the ratio of 1:10 weight by volume and the resulting homogenate filtered through cheesecloth.

Planting and inoculation of border rows: Seeds of BG90-2 were sown in plastic pots as border rows and the seedlings were mechanically inoculated with the viral suspension at three weeks after sowing. The inoculated seedlings metamorphosed to infected rows.

Planting of test cultivar and inoculation with vectors: Seeds of Moroberekan were planted two weeks after inoculating the border rows. Twenty plastic pots were used for sowing the rice cultivar. The experimental layout was a Complete Randomized Design (CRD) with three replicates. One week after planting the test cultivar, 80 adults each of non-viruliferous O. hyla and L. rubra were separately introduced onto the infected rows in each screenhouse to feed, acquire the virus and transmit to the rice plant.

In the experiment with mechanical inoculation, seedlings of Moroberekan were directly inoculated with the RYMV isolate at three weeks after sowing. Zero point five g of carborundum powder (600-mesh) was added to the inoculum to aid penetration of the virus into leaf tissues.

All recommended agronomic practices were carried out in both experiments.

Data collection: Data on disease severity and chlorophyll content were collected at 42, 56 and 70 days after sowing. These parameters were assessed on both the flag (youngest) and older leaves. The youngest leaf on each plant and three randomly selected old leaves per plant were evaluated for disease severity and chlorophyll content.

For disease severity, plants were scored based on a visual assessment of symptoms characteristic of RYMV infection on a Standard Evaluation System of 1, 3, 5, 7 and 9 according to IRRI (1996), where:

The chlorophyll contents of the leaves were measured using a SPAD 502 chlorophyll meter (Monje and Bugbee, 1992; Martines and Guiamet, 2004). SPAD-502 chlorophyll meter determines the relative amount of chlorophyll present in the leaves by measuring the absorbance of the leaf in two wavelength regions.

Data analysis: The percentage disease severity and chlorophyll content were calculated based on the visual scores and chlorophyll meter readings. Data on percentage disease severity and chlorophyll content were analysed using Statistical Analytical System (SAS, 2006). Mean separation was carried out using Duncan’s Multiple Range Test (DMRT) at 5% probability level.

RESULTS

There were considerable variations in percentage RYMV severity and chlorophyll content in the leaves of Moroberekan at the three physiological stages of assessment i.e., vegetative (42 DAS), flowering (56 DAS) and maturity (70 DAS).

In Table 1, there was a significantly (p<0.05) higher disease severity in the flag leaves of L. rubra inoculated plants at 42 DAS. The highest disease severity (59.05%) was observed in the flag leaves of mechanically inoculated plants at 42 DAS. There was no significant difference in disease severity in the old leaves of mechanically inoculated plant at 42 and 56 DAS. The lowest disease severity (34.64%) was recorded in the flag leaves of L. rubra inoculated plants at 70 DAS.

In Table 2, there was no significant difference in disease severity in the old leaves of mechanically inoculated plants at 56 and 70 DAS. The disease severities in the flag leaves of O. hyla inoculated plants were significantly different (p<0.05) at 42, 56 and 70 DAS. The highest disease severity (61.65%) was recorded in the old leaves of mechanically inoculated plants at 42 DAS while the last disease severity (22.97%) was noticed in the flag leaves of O. hyla inoculated plants at 70 DAS.

In Table 3, chlorophyll reduction varied from 20.25% in the flag leaves of L. rubra inoculated plants at 42 DAS to 49.34% in the old leaves of mechanically inoculated plants at 56 DAS.

Table 4 showed that there was no significant difference in chlorophyll reduction in the flag leaves of mechanically inoculated plants at 42 and 56 DAS. No significant difference was also noticed in the leaves (flag and old) of O. hyla inoculated plants at 42 and 56 DAS, as well as in the flag leaves of mechanically inoculated plants at 42 and 56 DAS.

Table 1:	Effect of plant age on percentage RYMV severity with mechanical and L. rubra inoculation methods
FLMC: Flag leaves of mechanically inoculated plants, FLLR: Flag leaves of L. rubra inoculated plants, OLMC: Old leaves of mechanically inoculated plants, OLLR: Old leaves of L. rubra inoculated plants and DAS: Days after sowing. In a row, means followed by a common letter are not significantly different at 5% level by Duncan’s multiple range test

Table 2:	Effect of plant age on percentage RYMV severity with mechanical and O. hyla inoculation methods
FLMC: Flag leaves of mechanically inoculated plants, FLOH: Flag leaves of O. hyla inoculated plants, OLMC: Old leaves of mechanically inoculated plants, OLOH: Old leaves of O. hyla inoculated plants and DAS: Days after sowing. In a row, means followed by a common letter are not significantly different at 5% level by Duncan’s multiple range test

Table 3:	Effect of plant age on percentage chlorophyll content with mechanical and L. rubra inoculation methods
FLMC: Flag leaves of mechanically inoculated plants, FLLR: Flag leaves of L. rubra inoculated plants, OLMC: Old leaves of mechanically inoculated plants, OLLR: Old leaves of L. rubra inoculated plants and DAS: Days after sowing. In a row, means followed by a common letter are not significantly different at 5% level by Duncan’s multiple range test

Table 4:	Effect of plant age on percentage chlorophyll content with mechanical and O. hyla inoculation methods
FLMC: Flag leaves of mechanically inoculated plants, FLOH: Flag leaves of O. hyla inoculated plants, OLMC: Old leaves of mechanically inoculated plants, OLOH: Old leaves of O. hyla inoculated plants, DAS: Days after sowing . In a row, means followed by a common letter are not significantly different at 5% level by Duncan’s multiple range test

DISCUSSION

This study revealed that RYMV severity and chlorophyll content of the leaves of Moroberekan are affected by age. Flag leaves were generally observed to be more susceptible to RYMV infection than older leaves. This observation corroborates the findings of Wamza et al. (2008) that reported the susceptibility of younger leaves of castor to leaf blight (Fusarium pallidoroseum) than older leaves, owing to more stomatal openings that permit pathogen penetration into the leaf tissues. The observation also agrees with the findings of Viruega and Trapero (2000) in which a negative correlation was reported between leaf age and disease severity in the development of olive leaf spot caused by Spilocaea oleagina. The result from this experiment is also consistent with the findings of Iqbal et al. (2002), who reported that two-week old seedlings of chickpea were more susceptible to blight (Ascochyta rabiei) than older plants. Similarly, the result is in agreement with the findings of Agrios (2005) who reported that plant age is important in disease infection, with younger plants being more susceptible.

Plants in their reaction (susceptibility or resistance) to viral diseases depend largely on age (Agrios, 2005). Host plants are susceptible mainly during the growing period and become resistant at maturity. Depending on the plant-pathogen combination, the age of the host plant at the time of arrival of the pathogen may considerably affect the development of infection and of an epidemic (Wamza et al., 2008). As plants get older, the tissues become more lignified, thereby making it difficult for pathogen to penetrate.

However, the results of this trial contradict the findings of Kurt and Tok (2006) who reported that the susceptibility of Parsley to Septoria blight (Septoria petroselini) infection increases with increasing leaf age. Findings from this study is also at variance with the observation of Ross (2007) who reported that lesion size on senescing leaf of African violets (Saintpaulia ionantha) infected with Corynespora leaf spot were comparatively larger in size than on mature leaves. In the same vein, Nelson (2006) reported that older leaves of tomato showed higher level of susceptibility to the late blight pathogen, Phytophthora infestans than the younger leaves. The observation from the present study also disagrees with the result of Goldgerg (2006) that reported higher susceptibility of older leaves of turfgrass to powdery mildew (Erysiphe graminis) than younger leaves.

Generally, there is a paucity of information in literature on why some plants become more susceptible to certain pathogens as they increase in age. However, Miller (1983) and Kus et al. (2002) attributed the scenario to a stronger biochemical and molecular interactions between the pathogen and the host plant with increasing leaf age. There are variations of opinion as to whether virus destroys chlorophyll or inhibits its synthesis. Sinha and Srivastava (2010) attributed the reduction in chlorophyll content of virus-infected leaves to the synthesis of chlorophyll, a compound that inhibits the development of chloroplast. Secretion of this compound is triggered by the interruption of the function of chloroplast in the tissues of chlorosis-induced plants. Lindenthal et al. (2005) also opined that the colonization and subsequent spread of a pathogen as the plant ages could weaken the structural organization of the host. This effect could increase susceptibility to infection in older leaves.

CONCLUSION

Moroberekan exhibited a high level of susceptibility to RYMV disease at the vegetative stage compared with maturity stage. Thus, infection by RYMV at the early stage would have a more devastating effect on the rice plant than at maturity. On the other hand, leaf chlorophyll content increased with age of the rice plant. Findings of this study showed that flag leaves of Moroberekan rice cultivar are more susceptible to RYMV infection than older leaves.

ACKNOWLEDGMENT

The authors are very grateful to the Government of Japan (Ministry of Foreign Affairs) for funding this research work. We also wish to acknowledge the technical support of Mr. Bayo Kehinde.