Effect of Sequence of Infection of Maize with Viruses Causing Maize Lethal Necrosis on Disease Development

Maize is the main staple food crop in Kenya. However, its production is constrained by maize lethal necrosis (MLN) disease, a result of dual infection of maize plants with Maize chlorotic mottle virus (MCMV) and a potyvirus Sugarcane mosaic virus (SCMV). In the field, infection by the two viruses is independent rather than occurring simultaneously since MCMV and SCMV are transmitted by different vectors. This study aimed at evaluating the effect of sequence of infection of maize by the two viruses causing MLN disease in the greenhouse. Two susceptible maize hybrids (DUMA 43 and H614D) were each infected with either of the two viruses first and later inoculated with the second virus. The plants were assessed for disease development weekly over a period of 2 months. Symptoms were significantly (p=0.05) more severe in maize plants initially infected with MCMV followed by SCMV, resulting in rapid death of plants compared to plants infected with SCMV followed by MCMV. The results indicate that severity of symptoms is influenced by the sequence of infection of maize plants with the causal agents of MLN disease. More remarkable was the synergism observed in maize plants pre-inoculated with SCMV followed by MCMV that had a slow manifestation of MLN disease symptoms. The results suggest that initial infection by SCMV may result in maize plants developing some levels of resistance resulting in initial suppression of MCMV infection. This study contributes to the understanding of resistance mechanisms exhibited by the plants during MLN disease development.

maize plants by the two viruses has any effect on symptom development and/or result in enhanced or reduced disease severity.

Virus Isolates used in the Study
The Sugarcane mosaic virus (SCMV) and Maize chlorotic mottle virus (MCMV) isolates used in the study were originally obtained from infected maize plants collected in Bomet County, in the South Rift-Valley Region of Kenya and propagated in susceptible maize variety H614D, which also served as the inoculum source. The isolates were maintained in separate secluded greenhouses to avoid contamination.

Experimental Design and Layout
The experiments were carried out in two cropping seasons, during the months of May to July and August to November 2016. Two maize varieties, H614D and Duma 43, which are extensively grown in most parts of Kenya, were used for the study. Pots, 45cm in diameter, were filled with sterilized soil to approximately three quarters full and mixed with Diammonium Phosphate (DAP 18:46:0) fertilizer at a rate of five grams per plant. The pots were watered to moisten the soil for ease in planting. Five seeds were planted in each pot at a depth of about 2.5 cm below the soil surface. The experimental design used was a completely randomized design (CRD) consisting of four replications per treatments and three plants in each replication. The pots were watered every morning and after emergence watering was done carefully every other day. The plants were thinned to three per pot at two leaves stage to ensure no overcrowding of plants during development. The plants were fertilized with calcium ammonium nitrate (CAN 26%) fertilizer at approximately 2.5 grams per plant at vegetative stage eight (V8). Virus inoculum for either of the viruses was prepared by grinding infected maize leaves with mortar and pestle in buffer in the ratio of 1:10 (w/v) of 0.1M phosphate buffer pH 7.0 (KH2PO4-4.8g, K2HPO4-10.8g, Na2SO3-1.26g in 1 liter of dH2O, pH7.4). Plant debris were removed by filtering the extracts through a muslin cloth. Each group of maize plants was inoculated with either SCMV or MCMV at V4 growth stage. After first inoculation with the initial virus, plants were allowed to grow for seven days before the second virus was introduced to have a combination sequence of SCMV followed with MCMV (SCMV + MCMV), MCMV followed with SCMV (MCMV + SCMV) to achieve MLN. Plants inoculated with the two viruses at the same time (MLN) and plants inoculated with a single virus were also established as controls.

Disease assessment and data analysis
Symptom severity was assessed and recorded based on a scale of 1-5 adopted from Gowda et al. (2015) where 1 = no symptom, 2 = <10% of plant leaf surface showing symptoms, 3 = 10-30% plant leaf surface showing symptoms, 4 = 30-50% of plant leaf surface showing symptoms, 5 = >50% of plant leaf surface showing symptoms. Analysis of variance (ANOVA) was used to determine the statistical significance of differences among treatments for the two viruses using GeneStat ® 2015 (v15.1) at 5% level of probability. Area Under Disease Progress Curve (AUDPC) scores were calculated for each virus and virus combination on individual plants using the midpoint rule method according to Campbell & Madden (1990) based on the formula: AUDPC = i=1 n-1 [(ti+1 -ti)(yi + yi+1)/2]; Where "t" is time in days of each reading, "y" is the percentage of affected foliage at each reading and "n" is the number of readings.

Disease incidence and severity
Similar trends in disease development were observed in both experiments (May -July and August -November 2016) were similar in the two maize varieties used in the study. Treatments consisting of SCMV alone, SCMV+MCMV, and MLN had symptom expression with a severity score of 2 within the first seven days post inoculation (dpi) in the two maize varieties ( Fig. 1 and Fig. 2). Treatments with MCMV alone or MCMV + SCMV had no symptom manifestation in the first 7 dpi. However, symptoms rapidly developed from mosaic to tissue necrosis with severe plant stunting and death 21 dpi for MLN and MCMV +SCMV with a severity score of 4.5 ( Fig. 1 and Fig. 2) while the single infections of MCMV and SCMV showed mild symptoms (Fig. 3). The first systemically infected leaves of co-infection became highly chlorotic at 9 dpi and developed necrotic areas at 10 dpi while the leaves of SCMV or MCMV single infection showed consistent mosaic or chlorotic symptom by 10 dpi. The systemic symptoms caused by multiple viruses were initially similar to those of single virus infections. At 14 dpi, a symptom rating score of 4 was recorded for MLN. At 18 dpi, symptoms observed in the MCMV + SCMV inoculated plants were significantly more severe than all other treatments except MLN. As of 30 dpi, the symptoms observed on maize plants initially infected with SCMV + MCMV were also significantly more severe than those for singly infected plants (Fig. 4). At 40 dpi, maize plants initially inoculated with MCMV followed by SCMV had more severe symptoms leading to a 'dead heart' (Fig. 5)  46 with SCMV followed by MCMV but less than for those inoculated with MLN ( Fig. 1 and Fig. 2).

Area under disease progress curve
The two maize genotypes used in this study had similar reaction to infection by either of the viruses or their combinations as indicated by the Area Under Disease Progress Curve (AUDPC) (Fig. 6). Maize plants inoculated with MLN at the V4 growth stage had the highest mean AUDPC value for the observation period was recorded at 137.7 and 139.4 for H614D and DUMA 43, respectively. The lowest mean AUDPC scores recorded was 86.61 in H614D and 88.1 in DUMA 43 for MCMV treatment. AUDPC scores for MCMV+SCMV and MLN were responsible for SCMV resistance (Xia et al. 2016). It is interesting that in MLN synergisms, SCMV is likely to possess important mechanism for enhanced infection by the co-infecting virus, MCMV, which is viewed as the primary disease behind MLN development. One of important proteins playing a significant role in the potyvirus infectivity is the HC-pro, which is multifunctional and possesses counter-defensive capacity to suppress the PTGS of the host. In the synergistic interaction with SCMV and MCMV, it is also likely that the accumulations of both MCMV and MCMV-derived siRNAs in maize that is reported to be increased remarkably compared to single infection implies that the presence of potyvirus was not only in favour of its own multiplication within the host but also catalyzing the multiplication of the partner co-infecting virus (Xia et al. 2016). In this case of synergism, the introduction of SCMV led to increased replication of MCMV hence the striking increase in symptom expression.
At the end of the experiment, mixed infections with MCMV and SCMV induced more severe symptoms than those observed in single viral infections regardless of the sequence of infection. Thus, there was no obvious difference in the expression level of the disease between SCMV+MCMV, MCMV+SCMV and MLN infected maize plants. Although there was a delay in the disease manifestation for the SCMV+MCMV infected plants but MLN disease was later observed in the co-infection.
In order to understand more on the interaction of maize plants with viruses causing MLN disease similar studies should be conducted-involving varieties that are resistance to either of the single viruses and see how they would respond to sequence of infection. In addition, it would be important to evaluate and understand the role of the individual viruses in the synergism.

Acknowledgments
This study was supported in part by grants from the Kenya Agricultural Productivity and Agribusiness Project (KAPAP) Collaborative Research Project and The Association for Strengthening Agricultural Research in Eastern and Central Africa (ASARECA) -MLND Project. Special thanks to the management of KALRO-Biotechnology Research Institute (BRI) Biotechnology Centre where the study was undertaken.