The study sought to evaluate the response of maize hybrids and inbred lines, categorized as resistant, tolerant, or susceptible to MLN, inoculated with either MCMV alone or in combination with SCMV, at different stages of crop growth. The impact of inoculation was assessed in terms of disease severity, grain yield, and the extent of contamination observed in seeds.
In both the plants inoculated with (a combination of MCMV and SCMV) MLN- and MCMV-inoculated experiments, the disease severity scores and AUDPC values for the hybrids and inbred lines were much higher at the early inoculated growth stages (V5 and V10) than at the later growth stages.
In an earlier study , in which maize plants were co-infected with the same combination as used in the present study (MCMV and SCMV) at the 3rd-leaf stage, severe chlorotic symptoms appeared on leaves 9 days post-inoculation (dpi) and developed necrotic areas at 10 days dpi, respectively. It was also observed that in the mixed infected leaf, the accumulated level of MCMV genomic RNAi was much higher and there was a significantly decreased level of SCMV RNA. The same study also reported that the expression levels of MCMV genomic RNAs and coat protein (CP) were higher in maize plants inoculated with the combination than in those inoculated with MCMV alone. It was also demonstrated in two earlier studies [22, 26] that HC-Pro, the silence suppressor encoded by potyviruses, could enhance the pathogenicity and accumulation of heterologous viruses.
The titres of MCMV were also much higher in plants inoculated with the combination than in those inoculated with MCMV alone, irrespective of their genetic background or the stage at which they had been inoculated, probably because of the synergistic effect of the combination, especially during the early stages of growth. Greater disease severity during the early growth stages led to greater yield losses in the susceptible entries than in either resistant or tolerant entries. However, in plants inoculated with the combination at their later growth stages, the titres of MCMV and SCMV as well as disease severity were much lower, with little impact on yield.
Understanding the host–pathogen–vector interaction is important especially in diseases caused by multiple viruses. Such interactions may be neutral or synergistic or antagonistic . In neutral interactions, accumulation of viruses or disease dynamics show little difference between joint infections and those by individual viruses, nor does the phenotype change significantly, whereas in synergistic interactions, joint infections result in higher titres and greater disease severity. Such synergy has been attributed to various factors including greater replication of the pathogens, enhanced ability to invade new plant tissue, or greater interference with the plant’s defences . Maize lethal necrosis is a typical example of a synergistic interaction involving MCMV and a potyvirus such as SCMV .
Disease severity scores, AUDPC values, and MCMV titres (ELISA) were much lower in the hybrids than in the inbred lines irrespective of the type of inoculum (single or combination). The hybrids certainly offered greater resistance than the inbred lines did to MCMV or SCMV or both and the resistance to the combination was greater (V14 to R1). In plants inoculated early, the higher titres of both MCMV and SCMV clearly showed the synergism. Early inoculation resulted in greater necrosis and little yield in the susceptible hybrids, whereas the resistant and the tolerant entries showed less chlorosis and no significant reduction in yield. Host–pathogen interaction changed depending on the stage of inoculation, the provenance (hybrids or inbred lines), and the inoculum (MCMV alone or together with SCMV), changes that may also be attributed to the host’s defence mechanism or to the combined effect of the interaction and the defences.
In MLN, titres of MCMV increased with infection with anyone of the potyviruses and resulted in greater chlorosis and greater economic losses with, symptoms of MCMV showing more chlorosis with increased at higher temperatures . However, interference with the plant’s defence mechanism by viral proteins may facilitate replication, intracellular movement, and spread within the plant of other viruses as well . Increased virulence can be predicted as the result of the competitive advantage of a virulent parasite because mixed infections can alter the host’s immunity and bring about phenotypic changes in the host. Thus, higher virulence in a mixed infection can be the consequence of competition for resources .
In hybrids, the titres of SCMV following inoculation with the combination were higher in plants inoculated early than in those inoculated later, and titres of SCMV were much lower than those of MCMV, indicating that the impact of infection by SCMV on hybrids and inbred lines is much lower in the presence of MCMV. This finding is consistent with the findings of earlier studies, which showed clearly that the expression level of MCMV genomic RNA and of CP was higher in maize plants infected by both SCMV and MCMV than in those infected by MCMV alone . The synergistic effect of the combined infection by SCMV and MCMV was especially apparent in plants inoculated early: MLN was more severe in those plants than in those inoculated at later. It is likely that in maize plants inoculated with a mix of MCMV and SCMV at later growth stages, the interaction tends to be closer to being neutral, as evident in the lower titres of SCMV, whereas in plants inoculated early the interaction tends to be closer to being synergistic. In another study, in maize plants co-infected with MCMV and either WSMV, MDMV, or SCMV, the titre of MCMV was 1.6- to 11-fold higher than that in plants infected with MCMV alone [7, 25; 29Xia et al., 2016). In the present study (Supplementary Table 5), titres of MCMV (as expressed in terms of OD values) were much higher (1.676–3.635) than those of SCMV (1.246–2.2425).
Little increase in the titres of MCMV was observed when those of SCMV (ELISA) were negative or below the threshold; the titres of MCMV increased when the OD value of SCMV ELISA was at a certain threshold, because the multiplication of SCMV was minimal or negative; MCMV alone cannot multiply exponentially unless SCMV is present at or beyond a certain minimum threshold. This also correlates with the percentage yield loss. Either SCMV may favour faster multiplication of MCMV or SCMV may make plants more susceptible when they are inoculated at an early stage. Earlier studies on co-infection by MCMV and SCMV showed increased accumulation of MCMV and virus-derived, small interfering RNAs (vsi RNAs) from MCMV .
In an earlier investigation on ultrastructural changes in infected maize leaf cells, it was observed that starch grains present in the chloroplasts in the cells co-infected by MCMV and SCMV were much smaller than those in MCMV-infected cells (Wang et al., 2017). It was suggested that photosynthesis in these cells was significantly affected. It was also found in the study that the mitochondria in the co-infected leaf cells were severely damaged much earlier than in the leaf cells infected by MCMV alone. Thus, systemic necrosis in MLN-affected plants was mainly due to disruption of chloroplast photosynthesis and mitochondrial respiration. In another study, it was noticed that in cells co-infected with MCMV and SCMV, the concentration of MCMV increased more than fivefold, whereas there was no difference in concentration of SCMV between co-infected and single-infected inoculated plants . From these observations in the present studies, we can deduce that MCMV infection alone can cause mild symptoms and that in cells co-infected with MCMV and SCMV, both the viruses accumulate in greater amounts.
Grain yield of MLN-resistant hybrids inoculated with the combination at early growth stages (V5 or V10) was negatively correlated with disease severity scores and AUDPC values, whereas the susceptible hybrids showed no such correlation. When inoculated later, the yields of resistant, tolerant, and susceptible hybrids were negatively correlated to disease severity and AUDPC values. Beyene et al.  observed during their genetic analysis of various tropical inbred lines for resistance to MLN that the combining ability estimates indicate the prevalence of additive gene action rather than of non-additive gene action. The grain yield of hybrids inoculated at later stages could be possibly protected by the defence mechanism. In another study of several lines in Hawaii, it was observed that the level of resistance varied widely, and it was suggested that MCMV resistance in maize is a quantitative trait . Host factors might decrease viral multiplication; however, this needs to be confirmed. In plants inoculated at later growth stages by the combination, symptoms were less severe and MCMV titres were lower than in those that had been inoculated early. Susceptible hybrids inoculated early recorded higher disease severity, greater AUDPC values, and much lower yield.
As expected, grain yield in resistant hybrids and inbred lines was higher than that in susceptible ones irrespective of the type of inoculation. The differences between resistant and susceptible types were more pronounced in plants inoculated at the vegetative stages (up to V10) than in those inoculated at V14, VT, or the reproductive stage (R1). This result is different from that seen in a study involving maize dwarf mosaic virus (MDMV), which reported higher losses in grain yield in plants inoculated at an advanced growth stage . Predicting the performance of hybrids from the performance of their parental inbred lines has been unsuccessful due to the masking of dominance effects
Seeds of both hybrids and inbred lines were contaminated with MCMV irrespective of the stage at which they were inoculated, irrespective of their level of resistance, and irrespective of the type of inoculum. Thus, contamination with MCMV appears to be unrelated to either the level of resistance or the growth stage.
In conclusion, the experiments in the present study further our understanding of the effect of genetic background and development stage on disease severity, grain yield, and seed contamination. The study also confirmed the synergistic effect of SCMV and MCMV co-infection on disease severity, titre values, and grain yield, because these parameters were affected more in plants inoculated with the combination than in those inoculated with MCMV alone. Inoculation at an early growth stage resulted in higher levels of both SCMV and MCMV, the result of greater synergy. The synergistic interaction of MCMV and SCMV affected the susceptible genotypes more severely, with higher chlorosis and necrosis and total loss of yield. As expected, the resistant hybrids and inbred lines showed were better able to withstand the pressure of the disease and recorded much lower yield loss. Inoculation at the early stages accentuated the differences between the hybrids and the inbred lines and also resulted in the highest disease severity and AUDPC values; this observation is important in evaluating the impact of MLN in the field. We found that disease severity, especially virus titre (content,) was not necessarily correlated to loss in grain yield, except when resistant and susceptible genotypes were compared; however, in tolerant genotypes, the differentiation could be more complicated. Thus, the use of MLN-resistant hybrids is a better option for protecting maize from the devastating effect of MLN on grain yield, as these hybrids can withstand disease pressure following infection even at early stages. The study also showed that MCMV titres were not affected either by the genotype or by the growth stage at which the plants had been inoculated. Therefore, there is a need to develop a seed treatment strategy to combat seed contamination of MCMV, particularly when maize is grown under conditions conducive to the virus.