Epidemiology, phytopathological and molecular differentiation and infection processes of diverse strains of Magnaporthe spp. on wheat and rice

Wheat blast (causal agent Magnaporthe grisea) is a novel and serious disease of wheat, causing high yield losses which has become a critical problem for wheat production in South America. This study aimed to improve the knowledge of characterization, epidemiology and pathogenicity of Magnaporthe spp., and to distinguish Magnaporthe grisea from wheat with another blast pathogen from rice (Oryza sativa L.), Magnaporthe oryzae in terms of phylogenetic relationships and interaction with its hosts and non-hosts. The results from this research would provide useful information for the development of strategies for the future control of wheat blast under practical field conditions. Initially, cultural characteristics of seventy Magnaporthe isolates were characterized in vitro. Diversity of colony morphology of these isolates was shown on V8 medium and significant differences in colony growth diameter and mycelial dry weight were demonstrated. These differences were significant among isolates from the same host, but insignificant between different hosts. Effects of temperature (20, 23, 26, 29 and 32°C) and spike-wetness periods (24h, 48h, 72h and 96h) on ear infection at flowering stage of wheat (BR 18) were studied. The results showed that higher temperatures (>26°C) are conducive for the growth of M. grisea. Differences in spike-wetness periods were insignificant, but disease severity increased in warm conditions with extended wetness periods. Therefore, the optimum inoculation conditions for further studies was 26°C and 24h wetness duration after inoculation. Pathogenicity tests were carried out in the greenhouse. Seventy Magnaporthe isolates collected from different host plants and geographical origins were inoculated on wheat and rice seedlings under controlled conditions to evaluate their infection capacity on wheat and rice leaves. All isolates produced typical lesions on their host plants, but there were significant differences in disease incidence and severity between the different host-isolate combinations. Thirty-six isolates from wheat and other grasses and eleven rice isolates were able to cause lesions with varying severity on both wheat and rice while the rest of the isolates were pathogenic only on their host plants. In the compatible interactions, 55% of the wheat isolates and 93.3% of the rice isolates successfully induced disease (severity between 4 to 5), which means the pathogen has developed on between 50% and 80% of the host leaves, and 32.5% of wheat isolates infected over 80% of wheat leaves. For the incompatible interactions, very low levels of disease severity were found on both host plants. Molecular research tools have also been developed and applied. Amplified fragment length polymorphism (AFLP) was used to detect genetic diversity within seventy isolates of Magnaporthe spp. from different hosts and geographical origins using three AFLP primer combinations. The isolates clustered into two groups corresponding to their original host. Magnaporthe grisea isolates from wheat were grouped together with 74% similarity, and resulted in the formation of two subgroups. Isolates from finger millet and perennial ryegrass were closely related to wheat isolates at a similarity level of 87.5%. Rice isolates (Magnaporthe oryzae) also clustered together with 79% similarity. However, isolates which originated from the same host but were collected from different countries were distributed in different subgroups or clades, instead of clustering together. The assessment of phylogenetic relationships using AFLP revealed that the variation assessed by genetic diversity studies within Magnaporthe spp. is mainly dependent on the host species, rather than the geographical origin. Furthermore, another molecular genotyping method, multilocus sequence typing (MLST), was established. A phylogenetic analysis among twenty Magnaporthe isolates from different hosts was performed by inferring dendrograms for the concatenated sequences of three home-keeping genes, actin, ß-tubulin and calmodulin. Due to the differences in mutation frequency, the concatenated sequences were divided into two parts, concatenated introns and concatenated exons, and their diversity was calculated separately. Dendrograms constructed using introns revealed three groups which contained the isolates from different hosts. Similar clusters were also found in the dendrogram derived from exons. The genetic diversity assessed by Nei`s parameters revealed that the percentage of polymorphic loci in the intron sequences was higher than that in the exon sequences. The diversity within the group, which differs depending on the geographical origin, was lower than that among four groups classified by their host plants. The clusters and genetic diversity produced by MLST suggested that in three house-keeping genes, most of the variation is explained by genetic diversity between host species rather than geographical origins, which is in accordance with the results of AFLP. In addition, a DNA based quantitative real-time polymerase chain reaction (PCR) was evaluated to quantify fungal biomass of Magnaporthe spp. in wheat and rice leaves. Samples from inoculated wheat and rice leaves were collected at different time points (0, 2, 4 and 6 dpi) and their Pot2 transposon was amplified with primers pfh2a and pfh2b. At the same time point, the amount of DNA biomass from the wheat isolate MG 31 infection on wheat leaves was higher than the other two isolates with their host plants. At 6 dpi, the biomass was 199 pg/mg for MG 31, 174 pg/mg for MG 5 and 133 pg/mg for Ca 89. For the non-host reaction, a very small increase of biomass was detected at 6 dpi in rice leaves infected by MG 31, but not in the two other incompatible interactions. Cytological investigations were performed with three Magnaporthe isolates selected from wheat (MG 5 and MG 31) and rice (Ca 89) for evaluating their capacity to infect the leaves of host and non-host plants. Microscopic observations were carried out by confocal laser scanning microscopy (CLSM) at different time points (12, 24, 48 and 72 hpi) and the growth of hyphae was classified into four stages. Spore germination and appressorium formation (stage one) were observed at 12 hpi in all interactions. At 24 hpi, only adapted isolates grown on their host were found and the percentages grown were 18%, 30% and 19% for isolates MG 5, MG 31 and Ca 89, respectively. At 48 hours post inoculation, some of the hyphae from adapted isolates (26% from MG 5, 29% from MG 31 and 27% from Ca 89) extensively grew in the primary invaded cells, and attempted to enter the adjacent cells. Multicellular infection (stage four) from isolate MG 5 (42%), MG 31(50%) and Ca 89 (32%) were observed at 72 hpi. For the invasion of adapted isolates, MG 31 appeared to grow more rapidly in its host plant than the other two isolates. The nonhost resistance was restricted in the interactions for rice isolate Ca 89 on wheat and for wheat isolate MG 5 on rice, but another wheat isolate MG 31 showed a certain degree of invasion in rice. In a parallel study, cellular responses of wheat (BR 18) and rice (CO 39) to both adapted and non-adapted isolates of the blast fungus Magnaporthe were investigated by autofluorescence microscopy at 48hpi and grouped into four response types. The observations revealed that most of the compatible interactions between adapted isolates and their host plants reached type D (41.3% for MG 5, 47.7% for MG 31 and 43% for Ca 89) at 48 hpi, when hyphae successfully occupied the whole invaded cell and even grew into the adjacent cells. Inoculation with non-adapted isolates resulted in a lack of effective penetration, and resistance was detected in wheat leaves against rice isolate Ca 89 (81.3%) and in rice leaves against the wheat isolate MG 5 (76.9%). Partial resistance occurred as a response in rice to isolate MG 31 derived from wheat where only 13.2% of hyphae from MG 31 were able to invade rice epidermal cells. The initial cellular defense towards the non-adapted isolates was associated with the formation of papillae (type B) and hypersensitivity responses (type C) which occurred in the initially invaded cells by showing strong autofluorescence and preventing further invasion from hyphae. Finally, a preliminary test on phytotoxins has been implemented. Detached seedling leaves of wheat and rice were treated with culture filtrate, infected leaf extract leachate and conidial suspension of Magnaporthe spp. as well as the filtrate of fresh potato dextrose broth (PDB) as a control. Symptoms were only evident on leaves treated with conidial suspension from compatible isolates. This result suggests that phytotoxins do not play a role in infection with three isolates (MG 5, MG 31 and Ca 89), neither on the seedling leaves of wheat nor on rice. Overall, the differences within Magnaporthe grisea isolates derived from wheat were evidenced in terms of phylogenetic characterization and pathogenicity. Genetic and cytological studies demonstrated differences between the two blast pathogens, Magnaporthe grisea (wheat) and Magnaporthe oryzae (rice). The results support the assumption that Magnaporthe grisea isolates (wheat) are distinct from those of rice, but some aggressive isolates may exhibit pathogenicity to rice. In order to further investigate the differences and relationships among Magnaporthe spp., more isolates should be collected from other hosts and geographical origins. In addition, research should also be expanded to practical field conditions and associated with the study of wheat blast management.