Evaluation of Maize Inbred Lines for Iranian maize mosaic virus (IMMV) Resistance

This work was carried out in collaboration between all authors. Author AE anchored the field study, gathered the initial data and performed preliminary data analysis. Authors BH and AD designed the study and wrote the proposal. Author KI edited the initial draft of manuscript and involved in technical supports. All authors read and approved the final manuscript. ABSTRACT In present study, the putative resistance capacity of thirty five maize inbred lines against Iranian maize mosaic virus (IMMV) was studied. Reaction to IMMV was analyzed under natural field infection and controlled conditions in a greenhouse in 2010 and 2011. In the greenhouse experiments, the maize plants were inoculated at two-leaf stage using the planthopper Laodelphax striatellus. In the field trials, an early sowing cultivation was used to facilitate a higher infection rate. The responses of inbreds to IMMV were assessed by symptom development and ELISA. The rate of natural infection of IMMV in susceptible control (SC704) was about 20%. MO17 showed about 40% infection in the first year. Both field and greenhouse results confirmed that MO17 was more susceptible to IMMV than SC704 and more reliable to be used as susceptible control in future studies of IMMV. Sowing one row of SC704 as vector spreader between every 5 rows of inbred lines caused sufficient vector propagation and virus transmission. Results of both field and greenhouse experiments showed little and no IMMV infection on K1263/1 and K3547/5, respectively. Hence, they were considered as IMMV-resistant. These lines with CIMMYT origin can also be used for production of resistant hybrids. Results showed that resistance to IMMV was not associated with maize maturity because resistance and susceptibility were found in both early and late matured inbred lines. Disease incidence and ELISA values were strongly correlated. Reduced plant height, ear weight and ear diameter and length and delayed silking were observed in plants infected with IMMV.

Rhabdoviruses are single stranded negative sense (non-coding) RNA viruses with a monopartite genome and five major structural proteins. Plant rhabdoviruses are transmitted by cicadellid leafhoppers, delphacid planthoppers and aphids in a persistent propagative manner [7][8][9]. The rhabdovirus maize mosaic virus (MMV) causes an important disease of maize in the tropical and subtropical regions in Africa, South America, Hawaii and Australia [2,5,10,11]. MMV is transmitted by the planthopper Peregrinus maidis [8].
IMMV is one of the most widespread causal agents threatening maize cultivation in temperate regions of Iran (28° to 31° N) [17]. IMMV is mainly transmitted by the planthopper Laodelphax striatellus under Iran conditions [12]. Climatic changes and favorable temperatures for the planthopper vector exacerbate the problem. A delayed sowing till mid June and use of chemicals, however, are known to reduce vector transmission [13]. Yet, these methods are costly and have low efficiency. Use of varietal resistance is a cost-effective, environmentalfriendly and convenient strategy to control plant viral diseases [14].
IMMV was first reported in 1979 from Fars province of Iran [15]. Despite some similarities to MMV, it appeared distinct in morphology, serology and biological characteristics [12,[15][16][17][18]. IMMV has become epidemic since 2003 in temperate regions of Fars, Iran. This was mainly related to the wide distribution of the vector [12]. Maize fields are usually infested by viruliferous L. striatellus populations when seeds are sown early in May [13,19,20]. Conversely, delayed cultivation limits the propagation and the growth of the vector and inhibits viral transmission. Although this strategy is useful, the best method to control viral diseases is breeding for varietal resistant.
Natural and/or artificial inoculations have been used for maize resistance evaluation against various viruses, such as sugarcane mosaic virus and maize dwarf mosaic virus [21], maize stripe virus and maize mosaic virus [22,23], maize streak virus [24], maize rayado fino virus [25], maize rough dwarf virus [13,26,27] and other viruses [28]. But this work is the first report of maize resistance to IMMV under field and greenhouse conditions. Different methods can be used to correctly diagnose plant diseases via symptoms. But precise diagnosis is very crucial when high numbers of genotypes are screened for resistance. ELISA is one of the most specific and easiest methods that provides rapid and precise virus detections [29][30][31]. ELISA is used to test a large number of genotypes in a relatively short period of time. Since its introduction by Clark and Adams [32], ELISA has accelerated the detection of viruses in plant materials, insect vectors, seeds, and vegetative propagules [32][33][34][35]. Therefore, accessing genetic diversity and using precise viral diagnosis methods are important for detection of varietal resistance.
The aim of this work was to investigate the responses of 35 maize inbred lines of Iranian maize breeding programs to IMMV using symptomatology and ELISA in greenhouse and field trials.

Field Experiments with Natural Infection
Thirty five maize inbred lines were subjected to viral infections (Table 1). These lines were supplied from Seed and Plant Improvement Institute, Karaj, Iran. The SC704 cultivar was used as a susceptible local check for IMMV [13]. Field experiments were carried out at Fars Research Center for Agriculture and Natural Resources, Zarghan station (29° 47' N, 52° 43' E), southern Iran, over the years 2010 and 2011.
Inbred lines were arranged in a randomized complete block design with three replications. Genotypes were sown in 5 m single rows 0.75 m apart. Two seeds per hill were sown and seedlings were thinned to one at the two-leaf stage. Final density was 20 plants per row spaced 25 cm apart. A single row of SC704 was sown between every five rows of inbred lines as viral spreader. The best sowing time for maize grain production is 1-10 th June in temperate regions of Fars [13]. However, to provide appropriate conditions for vector (L. striatellus) propagation and to enhance the rate of natural infection, seeds were sown on May 10 th . To supply the virus and its vector at the two-leaf stage, eight rows of SC704 were sown surrounding experimental plots on April 25 th . Weeds in the border and spreader rows were not controlled and no chemical was used. Results of the first year showed that MO17 is a more susceptible line to IMMV than SC704. Thus, both MO17 and SC704 were used as spreader in the field experiment in the second year to enhance the natural infection.
Symptoms of IMMV visually and clearly were detected 40-50 days after sowing, prior to the silking. In that time, all plants with IMMV symptoms were counted in each replication. The incidence of disease was estimated as the percentage of plants with symptoms. Traits such as plant height, leaf number, ear weight, ear diameter, ear length, cob percent (cob weight×100/ear weight) and day to silking were also recorded.

ELISA and Greenhouse Experiment
Greenhouse trial was performed at Plant Virology Research Center facilities, Shiraz University, Shiraz, Iran in 2011. Non-viruliferous vector planthopper (L. striatellus) was reared on barley. The planthoppers were placed on an infected maize plant for a week of acquisition feeding and transferred to susceptible barley plants for development of viruliferous colonies. Planthoppers from these colonies were used to inoculate maize seedling at two-leaf stage. Two seeds of each line were sown in a pot which were later thinned to one seedling per pot. The pots were individually kept in insect proof cages covered by nylon-mesh. Fifteen replications (pots) of each line were exposed to 14/10-h light/dark photoperiod. Inoculation of plants was carried out using 4-5 nymph viruliferous planthoppers at the two-leaf stage. Four weeks after artificial inoculation, samples of the newly emerged leaves were subjected to ELISA [35,36]. At this time the susceptible local check showed typical symptoms of IMMV. Leaf samples from healthy SC704 plants and their infected counterparts were used as negative and positive control, respectively. The alkaline phosphatase reactions were measured by determining absorbance at 405 nm with an MR 700 ELISA plate reader (Dynatech Laboratories, Chantilly, VA). Plants were considered ELISApositive for the virus presence if the mean A405 absorbance was higher than X +3SD in negative control where, X and SD denotes mean absorbance and standard deviation of the mean, respectively.

Statistical Analyses
The percentage of infected plants with IMMV in each plot of the field trials was considered as diseases incidence. Data were subjected to square root transformation, simple analysis of variance for each year and combined analysis of variance of two years using SAS software [37]. Biplot curves were drawn based on principal component analysis (PCA) to show the relationships between different traits and genotypes under natural infections and ELISA values using Genstat 12 th edition software. ELISA data were analyzed based on randomized completely unbalanced design procedure. Mean comparisons were carried out using Duncan multiple range test. ELISA negative genotypes that had significant difference with susceptible check were considered IMMV-resistant.

Appearance of Symptoms
Symptoms of IMMV began to appear on susceptible genotypes about 20 days after sowing and continued to develop until silking. IMMV early symptoms were chlorotic spots at the leaf base which developed as continuous/ discontinuous chlorotic stripes on leaves and sheaths, accompanied by stunting of the plants and abortion of ears (Fig. 1). The symptoms were similar to those reported earlier [15,38].

Field Experiments
Simple analysis of variance showed that genotypes had significant variations for the incidence of IMMV in each year ( Table 2). Incidence mean in the first year trial (9%) was higher than in the second year (6%). However, combined analysis showed that incidence rates in the two years were not significantly different (Table 3). IMMV incidence in some genotypes was higher than the local check, which means an extra source of IMMV inoculums was available for vector transmission.    [39]. Chen et al. [40] used disease incidence to show the response of maize inbred lines to maize rough dwarf virus (MRDV) under field conditions [40]. In their study, genotypes with 0 to 2% incidences were considered as highly resistant, while incidences of 10.1% to 20.0% and 20.1% to 100% were categorized as susceptible and highly susceptible, respectively.
Resistance to MMV has been previously reported in other studies [4,10]. Morphological, serological and molecular studies have confirmed that IMMV is distinct from MMV and other rhabdoviruses infecting gramineous plants [12,17,18,41]. Therefore, MMV resistance does not guarantee resistance against IMMV.
In the previous studies under natural infection conditions, maize genotypes displayed variable resistance to some viruses such as MRDV [26,[42][43][44]. Resistance to MRDV was detected under natural infection conditions in China [45][46][47]. Natural infection was also used for identification of resistant genotypes to MMV [10]. However, the present work is the first report of resistance against IMMV.
Identification of virus resistance generally involves inoculating a genetically diverse array of germplasm with virus and identifying plants that show either no or low infection. Most frequently, infection is scored by the percent of symptomatic plants. However, it is useful to ensure that the resistance is associated with reduced virus titer using serological or molecular techniques [5]. In this study, resistance and susceptibility of genotypes in the field were confirmed based on determination of virus titer in a greenhouse trial.

Greenhouse Experiments
Although some of inbred lines showed no IMMV symptoms under field conditions, their reaction to viral infection was tested by ELISA under artificial inoculation in a greenhouse trial. IMMV is not transmitted by mechanical inoculation [1]. Thus, an artificial inoculation of IMMV was performed by L. striatellus planthopper under greenhouse conditions.
Their OD values also were lower than 0.19. IMMV incidences of these inbred lines in field trials were lower than the susceptible genotypes (SC704 and MO17) which show their resistance to IMMV. Greenhouse trial confirmed field results, because K1263/1 and K3547/5 with no IMMV symptoms in the field had also low ELISA values. These two lines could be used as IMMV resistant for further genetic studies in breeding programs. The genotypes K3640/5, MO17, Oh43, A679, B73, L105, K722, K1728/8, K3615/2 and K18 are widely used in Iran in maize breeding programs but they are highly susceptible to IMMV. MO17 was the most susceptible genotype to IMMV. MO17 and B73 are the parental lines of SC704 hybrid, and have been reported as susceptible lines to other viruses [22,23,51,52].
Combined analysis of variance showed that infected and non-infected plants were significantly different for plant height, ear weight, ear diameter, ear length, cob percent and day to silking. Except cob percent and day to silking, infected plants had lower values for interested agronomic traits compared to non-infected plants ( Table 6). The significance of genotype effect for traits implies the existence of suitable genetic diversity among maize inbreds. Leaf number was not significantly different between infected and non-infected plants. Plant height reduction of infected plants is probably due to internodes reduction by IMMV. IMMV caused delaying in silking date. Plant height reduction and delaying in silking and kernel yield reduction from viral diseases has been reported earlier [12,13]. A study on MMV has indicated that maize plants can be dwarfed below 50 cm in height with no kernels produced under severe epiphytotics of the virus [53].  Principal component biplot showed the relationships of traits and genotypes (Fig. 2). Tight and acute angles between the vectors of OD and incidence show that they are strongly correlated. Ear weight (EW) strongly correlated with some ear characteristics such as ear diameter (ED) and ear length (EL) and its vector was in the same direction with leaf number vectors. The vectors of EL, ED and EW were in opposite direction with cob percent. This means that lower cob percent in ear causes higher kernel yield. Incidence and ELISA OD were related to reducing ear weight and other ear traits. Also longer day to silking seems related to higher incidence and higher OD. MO17 being in the vicinity of OD and incidence vectors shows its susceptibility to IMMV. In general, genotypes in right upper box in the vicinity of OD and incidence vectors are very susceptible to IMMV. K1263/1, S61 and TVA926 located in lower left box were far from OD and incidence vectors, which shows their resistance to IMMV (Fig. 2).  Table 1 4

. CONCLUSION
The present study was conducted to screen 35 maize inbred lines for their reaction to IMMV. This work is the first combined field and greenhouse evaluation of maize genotypes.
The data obtained proved resistance of K1263/1 and K3547/5 and susceptibility of MO17, K3640/5, Oh43, K3651/1, B73, L105, K722, K1728/8, K3615/2 and K3493/1 to IMMV. The genotypes K1263/1 and K3547/5 are the first Iranian IMMV-resistant inbreds with CIMMYT origin that can be used in production of resistant hybrids. K1263/1, K3547/5 and MO17 are genetically homozygos, which is a prerequisite for production of segregating populations and suitable for genetic studies via QTL analysis. K1263/1 is the parental line of an early maturing hybrid, SC260, known as the commercial cultivar Fajr [19]. This hybrid can probably be used in delayed sowing patterns for vector avoidance in temperate regions.
Plant height, ear weight and ear diameter and length were reduced in plants infected with IMMV, but cob percent increased and silking delayed in IMMV-infected plants. As this experiment is the first of its kind in Iran, it lays the foundation for further studies of maize viral diseases programs. Detected resistant lines could be used further as a check control genotype to confirm resistance of a genotype and incorporation in hybrid producing programs as parental lines.