Genetic diversity of the blast fungus, Magnaporthe grisea (Hebert) Barr, in Burkina Faso

Trapping nurseries trialed at two screening sites in Burkina Faso appeared to be an effective tool to characterize the virulence spectrum of blast populations using limited equipment and reduced labor. Itmade it possible to identify the best site to be used for screening for durable resistance. The effectiveness of some resistance genes indicated that they could be pyramided to provide durableresistance to blast fungus in Burkina Faso. The study also revealed the possible existence of new pathotypes in Burkina Faso. Fifty-five isolates of the blast fungus, Magnaporthe grisea, collected fromthe nurseries and rice fields were analyzed using random amplified polymorphic DNA (RAPD) PCR. Five major groups (Mg-1, Mg-2, Mg-3 Mg-4 and Mg-5) were defined. Mg-1, Mg-2 and Mg-3 were the largestgroups representing, 30.9, 25.5 and 30.9% of the 55 isolates analyzed. Only 9.1 and 3.6% belong to Mg-4 and Mg-5, respectively. Our results confirmed that RAPD PCR offers an inexpensive and speedy meansof generating markers for analyzing the population structure of the blast fungus

Genetic Variation Analysis of Mold (Magnaporthe oryzae B.Couch) Using Random Amplified Polymorphic DNA

Magnaporthe oryzae B.Couch is a host-specific fungi, certain strain only infect certain host plant species. Genetic variety among M. oryzae isolates was explained by dendogram which was constructed using similarity data of Random Amplified Polymorphic DNA (RAPD). Dendogram construction was achieved by computer software, Numerical Taxonomy System (NTSYS). The aim of the research were to study the genetic variation among M. Oryzae using RAPD and to construct a dendogram of genetic similarities among the ten isolates from green foxtail (Setaria viridis L.), finger millet (Eleusine coracana L.) and rice (Oryza sativa L.).RAPD was performed in 30 cycles using 5 primers (OPA-02, OPA-03, OPA-04, OPA-05, OPA-07). Polymorphism data was used to constructed dendogram using Dice index and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) in NTSYS software. There were 68 polymorphism fragments from 74 amplified fragments.Three clusters were formed in the dendrogram, based on host pathotype: foxtail millet type, finger millet type and rice type. There were two subclusters in foxtail millet type based on mating type, MAT1-1 dan MAT1-2. Thus, RAPD could be used as a method for genetic variation analysis of Magnaporthe oryzae to show host-specific specificity.Key words: Magnaporthe oryzae, RAPD, mating typ

Rice Blast Disease in the U.S. and Africa: Determination of Pathogen Diversity and the Identification of Resistance Genes for Disease Management

Rice blast caused by Magnaporthe oryzae (= Pyricularia oryzae) B. Couch, is a leading disease of rice. Magnaporthe oryzae exhibits a high degree of diversity. The diversity of isolates of M. oryzae from Africa and the U.S. were examined using vegetative compatibility and virulence phenotyping as well as determination of variation in the avirulence gene AVRPiz-t in isolates from Africa. Also, evaluation of blast resistance genes in the interspecific rice germplasm “New Rice for Africa” NERICA was done using F2 progeny of the cross of U.S. susceptible cultivar M204 and NERICA 12. The U.S. isolates were in three vegetative compatibility groups (VCGs), whereas isolates from Africa were in one to four VCGs that were unique to each country. Among isolates from the U.S, four pathotypes were distinguished based on differentials with the CO39 genetic background whereas 10 were distinguished based on the differentials with the LTH background. The R gene Pi9 was the most effective against isolates from both Africa and the U.S. Also, interspecific rice cultivars were highly resistant against isolates from both the U.S. and Africa. No single genotype or R gene was effective against all isolates from both Africa and the U.S. Blast control in the U.S could be achieved by introgression of Pi9 and Pi11 into rice cultivars. In West Africa, blast control could be achieved by introgressing Pi9, Pita2 and Pik-m into the rice cultivar F6-36. Alternatively, Pi9, Pik-s and Pik-m, could be introgressed into the rice cultivar FKR62N. For blast control in East Africa, one could introgress Pi9 into NERICA 12 or Pi9 and Piz-5 into NERICA 2. Variation in 10 of the 70 open reading frame (ORF) sequences of AVRPiz-t examined were observed. From the ORF sequences eleven haplotypes were observed with one haplotype comprising 86% of both virulent and avirulent isolates. Six virulent isolates had single nucleotide substitutions, insertions or deletions which altered the amino acid sequences of the ORF that could have caused isolates to be virulent. Based on PCR markers, two R genes, Pib and Pita2 were found in NERICA 12, but NERICA 12 could possibly contain other R genes

Genetic improvement of resistance to blast and bacterial blight of the elite maintainer line Rongfeng B in hybrid rice (Oryza sativa L.) by using marker-assisted selection

Rice blast caused by the fungus Magnaporthe grisea and bacterial blight (BB) caused by Xanthomonas oryzae pv. Oryzae (Xoo) are two major rice diseases in the world. An elite, early maturing maintainer line of hybrid rice, Rongfeng B hybrid rice is susceptible to both blast and BB. For improving its diseases resistance, BL122 and CBB23 were used as the donors of blast resistance genes Pi1 and Pi2 and BB resistance gene Xa23, respectively. These resistant genes were introgressed into Rongfeng B by using a marker-assisted backcross breeding programs, and two improved lines D521 and D524 with Pi1, Pi2 and Xa23 were developed. The results indicated that both improved lines showed high resistance to leaf and neck blast and BB. The resistance frequencies for the rice blast and the length of lesions resulting from BB ranged from 96.7 to 100% and 0.77 to 1.18 cm, respectively. The two improved lines showed the desired variation in the majority of evaluated agronomic traits, including the number of grains per panicle, the grains weight, plant height, and seed setting rate. A new cytoplasmic male sterile line, Rongfeng 3A, with Pi1, Pi2, and Xa23, was successfully developed through successive backcross breeding.Keywords: Gene pyramiding, marker-assisted backcross breeding, rice blast, bacterial bligh

Review of agronomic and genetic diversity of Moroccan rice varieties, and their resistance to blast disease (Pyricularia oryzae)

Received: October 25th, 2022 ; Accepted: January 25th, 2023 ; Published: March 9th, 2023 ; Correspondence: [email protected] study on agronomic and genetic characteristics of rice has given us scope to select varieties with desirable characteristics to mitigate various constraints. Rice (Oryza sativa) is the staple food for half of the world's population. However, its production is hampered by a variety of biological constraints. The Blast disease (Pyricularia oryzae) is an important rice disease, and one of the most effective control methods is to use resistant varieties. Study areas in Morocco include the Gharb plains. For all methods, cultural practises like soil levelling seem to be important, but biological control is not widely adopted due to cost, efficacy, and climatic conditions. The bibliographic synthesis was carried out in this context with the main goals of contributing to a better understanding of rice cultivation in Morocco; to identify and characterise the structure of the rice blast pathogen (Pyricularia oryzae), which will allow us to characterise the effects of rice blast; and to research on the Gharb rice field, which resulted in resistant varieties, which will potentially allow producers to have resistant varieties to overcome the diseases. The introduction and development of new rice varieties with high agronomic and socioeconomic value; the selection of lines with high yield, good grain quality, and precocity that are adapted to Moroccan conditions; as well as the development of new lines from Moroccan rice, are among the specific goals

“Epidemiology, virulence and molecular diversity in blast [Magnaporthe grisea (Hebert) Barr.] of pearl millet [Pennisetum glaucum (L.) R. Br.] and resistance in the host to diverse pathotypes”

Blast has emerged as an important disease in the major pearl millet growing areas in India. The present investigation was undertaken to study cultural, pathogenic and molecular diversity in the M. grisea isolates infecting pearl millet, effect of temperature and leaf wetness duration on blast development and to screen pearl millet lines for resistance to blast disease. Culture characters of four monoconidial isolates of M. grisea MgPM 45, MgPM 53, MgPM 56 and MgPM 118 were tested on OMA and PDA medium at different pH ranging from pH 5.5 to pH 8.0 (pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0) and different incubation temperatures 22oC, 24oC, 26oC, 28oC and 30oC to select optimum conditions for growth and sporulation of the fungus. Results of this study indicated that pH of 6.5 and temperature of 28oC is ideal for the growth and sporulation of M. grisea adapted to pearl millet. Based on these results, variability in the cultural and morphological characteristics of 65 isolates of M. grisea was studied on OMA medium at pH 6.5 by incubating at 28oC. Culture morphology varied significantly among isolates. A range of colour variation in the medium was also observed from buff colour to black among field isolates with smooth or rough margin. The radial growth of the M. grisea isolates varied significantly; maximum radial growth of 4.25 cm was recorded for isolates MgPM 125 and MgPM 162 whereas minimum radial growth of 2.30 cm was recorded for the pearl millet isolate MgPM 148. Large variation was also observed for sporulation among field isolates. It was observed that isolates with grayish black and brownish black growth with sector formation produced more spores. In majority of the isolates, maximum sporulation was confined to sectored region. These 65 isolates were also tested for pathogenic variation on a set of 10 host differentials (ICMB 93333, ICMB 95444, ICMB 97222-P1, ICMB 01333, ICMB 02444, ICMR 06444, 863B-P2, ICMR 06222 ICMR 11003 and IP 21187). The mean blast severity across the differentials was maximum for isolate MgPM 138 and minimum severity was observed for MgPM 132. Based on reaction type (avirulent/virulent), the 65 isolates were grouped into 28 different pathotypes. Pathotype G22 comprising isolates MgPM 121, MgPM 137, MgPM 138, MgPM 145, MgPM 148 from Rajasthan and MgPM 173 and MgPM 174 from Uttar Pradesh appeared as most virulent as it could infect all the 10 host differentials whereas pathotype G2 comprising MgPM 127, MgPM 129, MgPM 132, MgPM 149, MgPM 158, MgPM 159 and MgPM 39 was least virulent. These isolates could be grouped in five main clusters based on the results of molecular diversity study using URP markers. Among them cluster I (32) and III (31) included more than 95 per cent isolates whereas cluster II, IV and V contained 2-3 isolates. Studies on the effect of leaf wetness duration on disease development showed an overall increase in leaf blast severity, lesion length (mm), number of lesions per plant, lesion sporulation and leaf sporulation with the increase in leaf wetness duration (LWD). Based on the results it can be concluded, that both leaf wetness duration and temperature were essential for blast on pearl millet which becomes more severe at longer wetness durations beyond 48 hours during optimum day/night with a temperature ranging from 25±1/20±1oC to 30±1/22±1oC. For the identification of blast resistance, 160 designated B-lines of pearl millet were screened under greenhouse conditions against five pathotype-isolates viz., MgPM 45, MgPM 53, MgPM 56, MgPM 118 and MgPM 119. Multiple-pathotype (3-5) resistance was found in 23 lines. Eight lines (81B, ICMB 88004, ICMB 92444, ICMB 97222-P1, ICMB 02111, ICMB 07111, ICMB 09333 and ICMB 09999) were found resistant to all the five pathotypes. Similarly for the identification of stable sources of adult plant resistance, 28 lines were evaluated in the disease nursery (PMBVN) at six locations, Aurangabad, Dhule, Durgapura, Gwalior, Jamnagar and Patancheru during 2013 and 2014. None of the entries in the blast nursery was resistant at all the test locations. However, ICMR 06444 was found resistant at three (Gwalior, Jamnagar and Patancheru) locations and showed moderate resistance at other three locations. ICMB 01333, ICMR 11009 and HHB 146 improved (a hybrid) were resistant at Gwalior and Jamnagar. Pearl millet lines identified in this study that are resistant at 2-3 locations can be selected for use in pearl millet breeding programs aiming to develop blast resistant hybrids

“Epidemiology, virulence and molecular diversity in blast [Magnaporthe grisea (Hebert) Barr.] of pearl millet [Pennisetum glaucum (L.) R. Br.] and resistance in the host to diverse pathotypes”

Blast has emerged as an important disease in the major pearl millet growing areas in India. The present investigation was undertaken to study cultural, pathogenic and molecular diversity in the M. grisea isolates infecting pearl millet, effect of temperature and leaf wetness duration on blast development and to screen pearl millet lines for resistance to blast disease. Culture characters of four monoconidial isolates of M. grisea MgPM 45, MgPM 53, MgPM 56 and MgPM 118 were tested on OMA and PDA medium at different pH ranging from pH 5.5 to pH 8.0 (pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0) and different incubation temperatures 22oC, 24oC, 26oC, 28oC and 30oC to select optimum conditions for growth and sporulation of the fungus. Results of this study indicated that pH of 6.5 and temperature of 28oC is ideal for the growth and sporulation of M. grisea adapted to pearl millet. Based on these results, variability in the cultural and morphological characteristics of 65 isolates of M. grisea was studied on OMA medium at pH 6.5 by incubating at 28oC. Culture morphology varied significantly among isolates. A range of colour variation in the medium was also observed from buff colour to black among field isolates with smooth or rough margin. The radial growth of the M. grisea isolates varied significantly; maximum radial growth of 4.25 cm was recorded for isolates MgPM 125 and MgPM 162 whereas minimum radial growth of 2.30 cm was recorded for the pearl millet isolate MgPM 148. Large variation was also observed for sporulation among field isolates. It was observed that isolates with grayish black and brownish black growth with sector formation produced more spores. In majority of the isolates, maximum sporulation was confined to sectored region. These 65 isolates were also tested for pathogenic variation on a set of 10 host differentials (ICMB 93333, ICMB 95444, ICMB 97222-P1, ICMB 01333, ICMB 02444, ICMR 06444, 863B-P2, ICMR 06222 ICMR 11003 and IP 21187). The mean blast severity across the differentials was maximum for isolate MgPM 138 and minimum severity was observed for MgPM 132. Based on reaction type (avirulent/virulent), the 65 isolates were grouped into 28 different pathotypes. Pathotype G22 comprising isolates MgPM 121, MgPM 137, MgPM 138, MgPM 145, MgPM 148 from Rajasthan and MgPM 173 and MgPM 174 from Uttar Pradesh appeared as most virulent as it could infect all the 10 host differentials whereas pathotype G2 comprising MgPM 127, MgPM 129, MgPM 132, MgPM 149, MgPM 158, MgPM 159 and MgPM 39 was least virulent. These isolates could be grouped in five main clusters based on the results of molecular diversity study using URP markers. Among them cluster I (32) and III (31) included more than 95 per cent isolates whereas cluster II, IV and V contained 2-3 isolates. Studies on the effect of leaf wetness duration on disease development showed an overall increase in leaf blast severity, lesion length (mm), number of lesions per plant, lesion sporulation and leaf sporulation with the increase in leaf wetness duration (LWD). Based on the results it can be concluded, that both leaf wetness duration and temperature were essential for blast on pearl millet which becomes more severe at longer wetness durations beyond 48 hours during optimum day/night with a temperature ranging from 25±1/20±1oC to 30±1/22±1oC. For the identification of blast resistance, 160 designated B-lines of pearl millet were screened under greenhouse conditions against five pathotype-isolates viz., MgPM 45, MgPM 53, MgPM 56, MgPM 118 and MgPM 119. Multiple-pathotype (3-5) resistance was found in 23 lines. Eight lines (81B, ICMB 88004, ICMB 92444, ICMB 97222-P1, ICMB 02111, ICMB 07111, ICMB 09333 and ICMB 09999) were found resistant to all the five pathotypes. Similarly for the identification of stable sources of adult plant resistance, 28 lines were evaluated in the disease nursery (PMBVN) at six locations, Aurangabad, Dhule, Durgapura, Gwalior, Jamnagar and Patancheru during 2013 and 2014. None of the entries in the blast nursery was resistant at all the test locations. However, ICMR 06444 was found resistant at three (Gwalior, Jamnagar and Patancheru) locations and showed moderate resistance at other three locations. ICMB 01333, ICMR 11009 and HHB 146 improved (a hybrid) were resistant at Gwalior and Jamnagar. Pearl millet lines identified in this study that are resistant at 2-3 locations can be selected for use in pearl millet breeding programs aiming to develop blast resistant hybrids

Population analysis of the finger millet blast pathogen Magnaporthe oryzae in Eastern Africa

A thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of doctor of Philosophy.The main aims of the investigation were to develop an in-depth understanding of the genetic diversity, population structure and evolutionary relationships as well as to assess the sexual reproductive capability of the finger millet blast (FMB) pathogen Magnaporthe oryzae in Eastern Africa. A set of 300 M. oryzae isolates collected during 2000 – 2017 from key finger millet growing districts in Kenya, Uganda, Tanzania and Ethiopia were utilised in this study. Two novel molecular markers designated HyP1 and HyP2 were developed in this study and two known phylogenetic markers ITS (internal transcribed spacer) and HIS4 (histone 4 gene) were identified by bioinformatic analysis. Single- and multi-locus analysis provided a clear assessment of the FMB pathogen genotype diversity and distribution pattern. At the regional level in Eastern Africa, ITS and HIS4 revealed 7 - 9 genotypes, whereas HyP1 and HyP2 identified 80 - 85 genotypes reflecting their high resolution. Multi-locus sequence (MLS) analysis revealed 207 genotypes displaying a continuous genetic variation pattern of the FMB pathogen populations in Eastern Africa. Bayesian and reticulate network analyses distinguished the vast majority of genotypes into two sub-populations (designated as Group A and B), which were geographically clustered. Diagnostic PCR revealed the presence of a high proportion of M. oryzae isolates containing the Grasshopper (grh) repeat element in Ethiopia and Tanzania (e.g. 85 %). Reference genome assemblies have been established for two M. oryzae isolates representing the sub-populations identified. Genome resequence data has been developed for sixteen isolates representing the genotype diversity. Comparative analysis provided novel insights into the genomic architecture and evolutionary relationships in the FMB pathogen. Genomic regions and/or genes, putatively isolate specific have been identified. Phylogenomic analysis revealed monophyletic nature of the FMB pathogen in Eastern Africa and Asia suggesting a common origin. Genome-wide single nucleotide polymorphism (SNPs) ranges broadly corresponded to the sub-populations identified. Complete grh sequence has been defined and the presence of at least two versions of the element in the FMB pathogen in Eastern Africa has been shown. Mating type specific PCR assay revealed high proportions of the two mating types MAT 1-1 (56 %) and MAT 1-2 (44 %) in the contemporary population of the FMB pathogen in Eastern Africa and also in the four countries surveyed, albeit at variable levels. Mating culture assays established a high proportion of fertile isolates (60 %) and revealed the dominance of male sexual behaviour followed by hermaphrodite and female isolates. The emerging pattern is indicative of a decrease in the fertility status as well as the level of hermaphrodites and females. Integrated assessment of the mating type and fertility data along with the high genotype diversity and their continuous variation pattern observed is strongly suggestive of a mixed reproductive behaviour including episodic sexual reproduction. The new knowledge and resources generated contribute to the advancement of current understanding of the finger millet blast pathogen biology providing a framework for the effective utilization of host resistance in Eastern Africa as well as a strong platform for further research advances in the field

Plant pathogenic Magnaporthales in Australia, with particular reference to Pyricularia oryzae on wild and cultivated rice

The Magnaporthales is an order of fungi that contains plant pathogens and saprobes. This order consists of three families, Pyriculariaceae, Magnaporthaceae and Ophioceraceae, which are phylogenetically, morphologically and ecologically distinct. To date, about 200 species have been described in Magnaporthales, of which approximately 50% are plant pathogens. Some species are important pathogens of grasses and cereals such as the rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) and the take-all pathogen of cereals Gaeumannomyces graminis. The study of classification and identification of Magnaporthales in Australia and pathogenicity of Pyricularia oryzae are reported in this thesis