Bacterial Leaf Blight Resistance Genes Research Articles

Introgression of rice blast and bacterial leaf blight resistance genes into HPR2143 rice variety through marker assisted selection and Anther culture

The use of resistant rice cultivars is the most proficient way to overcome rice blast and bacterial blight diseases in rice. Cultivars, however, experience a rapid breakdown in their resistance, predominantly due to the advent of new pathotypes. Therefore, for durable resistance, it is important to combine multiple genes that impart resistance to more than one pathotypes into one genetic background. Foreground selection of BC2F2 plants of two crosses (HPR2143/DHMAS164 and HPR2143/PB1) was done to detect the presence of blast resistance genes i.e. Pita and Pi9 in these plants. Plants homozygous for gene Pita were screened by a pair of gene specific markers YL87/155 (coupling dominant) and YL87/183 (repulsion dominant). Likewise, homozygous positive plants for gene Pi9 were identified using the co-dominant marker Pi9STS-1. For bacterial blight (BLB) resistance genes, BC2F2 plants of two crosses (HPR2143/IRBB54 and HPR2143/PR114) were screened for genes Xa21 and Xa38 using gene-derived markers BLB248 and Oso4g53050-1, respectively. Anther culture of developed F1s was attempted for fixation of genes.

Recovery of Recurrent Parent Genome in a Marker-Assisted Backcrossing Against Rice Blast and Blight Infections Using Functional Markers and SSRs.

The most vital aspect of marker-assisted backcross breeding is the recurrent parent genome recovery. This enables the selection of only parents with recovered recipient/recurrent parent genome in addition to the targeted genes. The recurrent parent genome recovery (RPGR) ensures that non-desirable genomic segments are removed while the gene of interest is sustained in the recombined progenies without further segregations. This work was aimed at quantifying the RPGR of backcross populations with introgression of bacterial leaf blight resistance genes. Putra-1, a Malaysian elite variety, high yielding with inherent resistance to blast but susceptible to bacterial leaf blight (BLB), was crossed with IRBB60 which is resistant to BLB disease. The IRBB60 has four Xoo resistance genes—Xa4, xa5, xa13 and Xa21. Tightly linked polymorphic functional and SSR markers were used for foreground selection at every stage of backcrossing to select progenies with introgressed target genes. Background selection was done to quantify the percentage of RPGR in the selected lines using 79 confirmed polymorphic microsatellites. Result obtained showed that the percentage of RPGR was 80.11% at BC1F1, 95.30% at BC2F1 and 95.9% at BC2F2. The introgression of Xa4, xa5, xa13 and Xa21 Xoo resistance genes were faster through the adopted marker-assisted backcross breeding compared to what could be obtained through conventional breeding. All the 16 selected lines displayed resistance to BLB with three lines showing high resistance to the disease. The blast resistance contained in the genetic background of Putra-1 was also sustained in all the selected lines. The newly developed lines were recommended as new rice varieties for commercial cultivation.

Assessment of Bacterial Leaf Blight Disease of Indonesian Rice Germplasms Using SSR Markers

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious disease in rice plants worldwide. Yield losses caused by Xoo can be as high as 50% in some parts of Asia. Xa7 gene can potentially confer a broad resistance to BLB. Evaluation of disease resistance characteristics in early breeding generations of rice is important to develop varieties with better resistance. This study reports the evaluation of 167 Indonesian rice germplasms against three BLB isolates/pathotypes in a green house setting and the genotyping of 56 Indonesian rice germplasm using 12 SSR markers linked to Xa7 BLB resistance gene. The majority of the indigenous rice germplasms was found to be susceptible to three BLB isolates/pathotypes tested. Sate Liko from Bantul, Yogyakarta, Horeg from Cirebon, West Java and Sijem from Malang, East Java revealed consistent resistance to three isolates/pathotypes tested based on BLB evaluation in a greenhouse, UPGMA analysis, and genotyping. Pathotype XII displayed more virulence to Indonesian rice varieties tested compared to pathotypes VI and V. The association analysis using the General linear model identified six markers associated with BLB resistance and two markers were highly associated (RM20589 and RM20590). This information will be useful for future studies of BLB resistance in rice plants.

Zinc enriched Pseudomonas fluorescens triggered defense response in rice against bacterial leaf blight

Bacterial leaf blight (BLB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease in rice and Pseudomonas fluorescens is known as a successful bioagent against the pathogen. The present investigation was carried out to study the effect of micronutrients, viz. boron (B), iron (Fe), zinc (Zn), and molybdenum (Mo) on the efficacy of the bioagent and induction of defense mechanism in rice. The study revealed that P. fluorescens strain PfAs1 enriched with 50 ppm Zn could suppress the blight pathogen by 45.92% compared to control and other micronutrients under in-vitro condition. The results also demonstrated a reduction of 82.99% disease intensity in rice due to application of Zn enriched PfAs1. Further study was made to understand whether the Zn enriched PfAs1 (Zn PfAs1) induces defense response in rice. The levels of expression of defense related genes, viz. OSPR1a, OSPR1b, OSPR10a, Xa1 and Xa26 against Xoo was quantified by quantitative PCR (qPCR). During the interaction of rice with Zn PfAs1, a 20 fold increase in the expression of rice BLB resistance gene Xa26 and pathogenesis related (PR) gene OsPR1b, was observed when compared with controls. Similarly, 14 and 10 fold increase in levels of expression of OsPR1a and OsPR10a genes was recorded, respectively. An 8 fold increase in the level of expression of Xa1 gene was also recorded in the study. This is the first report on enhanced defense response in rice due to the antagonistic potential of Zn enriched P. fluorescens strain against BLB pathogen.

The genetic associations between yield component traits and bacterial leaf blight resistance in rice (O. sativa L.)

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is highly destructive to high-yielding susceptible rice cultivars. In severe epidemics, yield losses up to 75% have been reported. Considerable information on BLB resistant genes is available; however, genetic overlaps between BLB resistant and yield related traits largely remain unclear. Using two sets of backcross introgression inbred lines and one set of recombinant inbred lines, genetic associations between bacterial leaf blight resistance and yield component traits in three Xoo races were analyzed using Single Nucleotide polymorphism (SNP) markers. Fifteen quantitative trait loci conferring BLB resistant and 112 QTLs for yield and yield related traits were detected. Among 15 QTLs conferring bacteria leaf blight resistance, 14 QTLs overlapped with 39 QTLs for yield and yield related traits. The total resistance variation explained by additive QTLs (R2) ranged between 5-76% and epistasis QTLs ranged between 1-26%. Quantitative trait loci (QTLs) with the highest additive effects for BLB resistance were detected at positions os04-17305294 (on chr.4), os05-07310209 (on chr.5), os09-15240130 (on chr.9), os11-28421947 (on chr.11) and os12-25291547(on chr.12). The BLB resistance QTL at position os05-07310209 (on chr.5) overlapped with QTLs for 1000-grain weight, grain number per panicle, days to heading, grain length width ratio and grain yield per plant. The BLB resistance QTL at position os09-15240130 (on chr.9) overlapped with QTLs for grain yield per plant, panicle number, grain width and 1000-grain weight. The BLB resistance QTL at position os12-25291547-(on chr.12) overlapped with QTLs for 1000-grain weight, panicle number, grain yield per plant, filled grain number and grain width. Eleven percent of the 717 plants in this study were resistant to at least two Xoo races and had higher grain yield compared to both recurrent parents. Overlapping regions especially with already cloned genes and epistasis detected in this study offer opportunity to develop rice varieties which combine high yield and resistance to bacteria leaf blight.

Research Article Identification of bacterial leaf blight resistance genes in Malaysian local rice varieties

Research Article Identification of bacterial leaf blight resistance genes in Malaysian local rice varieties

Evaluation of Backcrossed Pyramiding Lines of the Yield-related Gene and the Bacterial Leaf Blight Resistant Genes

Evaluation of Backcrossed Pyramiding Lines of the Yield-related Gene and the Bacterial Leaf Blight Resistant Genes

Genetic analysis of microsatellites associated with resistance against bacterial leaf blight and blast diseases of rice (Oryza sativa L.)

Bacterial leaf blight (BLB) and blast are among the most destructive diseases of rice. In order to develop new varieties that can resist various races of the pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, which cause these two diseases, respectively, it is important to identify suitable markers and a marker-aided selection strategy. Seventeen foreground microsatellites associated with resistance against BLB and blast were screened for polymorphism for possible adoption in a breeding program for resistant cultivar development. A total of 472 background simple sequence repeats (SSRs) were also screened for polymorphism in order to recover the recurrent parent genome. All 72 F1 progenies were also analysed with the 17 foreground and 472 background markers in comparison to the alleles of their parents. The markers were validated using 218 BC2F2 plants. The result revealed six tightly linked polymorphic foreground markers (Xa21FR, pTA248, Xa13prom, MP, RM21 and RM13) associated with resistance against Xoo pathotype P7.7 and two microsatellites (RM6836 and RM8225) associated with resistance against blast pathotype P7.2. A total of 79 polymorphic background markers were identified for recovering the recurrent parent genome. The result also showed that the markers had goodness of fit (blast resistance) to 1:2:1 ratio for single gene model in BC2F2 progenies. These genetically confirmed microsatellite markers were recommended for use in marker-assisted backcross breeding and other marker-assisted selection of BLB and blast resistance genes in order to develop varieties with durable and broad spectrum resistance against the two major rice diseases.

Basmati rice lines development carrying multiple bacterial blight resistance genes pyramided using the marker-assisted backcross breeding approach

Basmati rice is treated as one of the world’s best rice due to its extra-long grain and unique aroma,. Over the last decade, Pakistan has earned more than US$2 billion through basmati export. Unfortunately, basmati rice is being threatened with bacterial leaf blight (BLB) disease that leads to poor production and quality. We attempted pyramiding of BLB genes through marker-assisted backcross breeding using Super Basmati as the recipient and IRBB60 as the donor parent. BLB resistance genes Xa4, xa5, and Xa21 were used for introgression. Molecular markers MP1 and MP2, RM122, and pTA248 were used for Xa4, xa5, and Xa21 genes respectively for foreground selection. The 85 SSR polymorphic molecular markers were used for background selection, which showed a more than 90% recurrent parent genome recovery. Phenotypic traits for grain dimensions were also considered while selecting backcross female parents from BC1F1 to BC5F1, which resulted in saving the resources from background selection before the selfing of backcrossed lines. Multiple lines carrying BLB resistance genes combinations Xa4 + xa5 and Xa4 + xa5 + Xa21 were developed. The BC5F4 uniform lines showed resistance against BLB disease at hotspot locations. One of the developed lines, PKBB15–116, which has a yield advantage of more than 10%, tolerant to lodging, and has basmati quality. These pyramided lines in basmati background will also be helpful for breeders to incorporate resistance genes in a shorter period of time, in order to develop new basmati rice varieties in future.

English

Bacterial leaf blight (BLB) of rice is one of the most destructive diseases affecting rice fields. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of BLB. Two BLB resistance genes, Xa21 and Xa7, were transferred into the susceptible indica cultivar Bacthom7 (BT7) by using marker-assisted selection with markers pTA248 for Xa21 and ID7 for Xa7. Improved BT7 lines carrying the two resistance genes were inoculated with three isolates of the Xoo from Northern Vietnam and evaluated for agronomic traits. Artificial inoculation of 13 lines with three Xoo races identified nine highly resistant lines with wide-spectrum resistance to Xoo, including D1, D2, D3, D6, D7, D8, D9, D10 and D12. These lines were similar to recurrent parent BT7 with regard to external appearance, yield performance and grain quality. On the basis of agronomic traits and the level of resistance to BLB, two promising lines, D6 and D9 were further selected. These two lines could efficiently contribute to rice production for food security and food safety in northern Vietnam.   Key words: Xanthomonas oryzae pv. oryzae, resistance genes, near-isogenic lines, marker-assisted selection (MAS), improved rice lines.&nbsp

Detection of Bacterial Leaf Blight Resistance Genes in Indigenous Glutinous Rice Landraces

Bacterial leaf blight disease (BLB) caused by Xanthomonas oryzae pv is one of the most widespread devastating diseases of rice worldwide. In this study, a total of 86 indigenous glutinous rice landraces were examined for BLB resistant genes included Xa4, xa5, and Xa7 by using molecular markers. The results indicated that 37 samples carrying resistant genes, in which Xa4 was found in 11 samples, xa5 determined in 6 samples, Xa7 was in 19 samples, respectively. However, both of Xa7 and Xa4 were observed in only one sample. The resistant level against 10 bacterial strains carrying the resistant genes was also evaluated. We found that the number 6, 9, and 8 out of the 10 bacterial strains were resistant to the landraces which carried the Xa4, xa5, and Xa7. The bacterial strain number 5 was revealed highly toxic, causing infection of all samples. The agronomical traits, yield and yield components of 37 rice landraces included the resistant genes (Xa4, xa5 and Xa7) were evaluated. Our findings may provide useful genetic sources in indigenous glutinous rice landraces to further develop BLB resistant rice lines via molecular breeding program.

Pyramiding of Three Bacterial Blight Resistance Genes in Rice Cultivar Using Marker Assisted Selection

Bacterial leaf blight (BLB) is a major disease of rice, limiting production throughout the world. Xanthomonas oryzae pv.oryzae is highly virulent pathogen, causing major losses in Rice. Present scenario needs development of disease resistant variety through marker assisted selection is the most promising way to improve their performance. Safri-17 is high yielding most popular rice variety of Chhattisgarh but it is highly susceptible to BLB disease. In the present investigation molecular markers were used to introgressed three major BLB resistance genes i.e xa5, xa13 and Xa21 into Safri-17 from a donor line RP-Bio226. Genetic analysis was used to confirm the F2 population for the presence of BLB resistance genes with the help of SSR and STS markers (RM6765, RM13, and pTA248, xa5R/S respectively). Breeding lines with two or three resistance genes were developed and tested for resistance to the BLB. They showed a wider spectrum and a higher level of resistance as compared to lines with only a single gene. The BLB resistance genes in homozygous condition (Xa21Xa21/xa13xa13/xa5xa5) showed resistance disease reaction whereas in heterozygous condition (Xa21xa21/Xa13xa13/Xa5xa5) showed moderate resistance reaction. Similarly two gene combinations gave more resistance reactions as compare to progenies have single gene.

Grain and Cooking Quality Characteristics of BB Pyramided Genotypes Derived from Cross Between CSR-30 and Pusa Basmati-1460

Grain and cooking quality traits are extremely important for Basmati rice in view of its culinary characteristics. A total of twenty-five bacterial leaf blight (BB) resistant genes (xa13 and Xa21) pyramided BC1F1 genotypes from a cross between CSR-30 and Pusa Basmati-1460 with high yield were evaluated for grain and cooking quality traits. Quality parameters measured were, kernel dimensions before and after cooking, aroma, alkali spread value (ASV) and amylose content. Based on agronomic evaluation, BB reaction and percentage recovery of recurrent parent genome (RPG), BC1F1 genotypes P–C-70, P–C123, P–C-142 and P–C-149 were found promising and were stringently evaluated for quality. Elongation ratio for CSR-30 was recorded 1.93. Elongation ratio among pyramided BC1F1 genotypes varied between 1.34 (P–C-142) to 1.91 (P–C-149). The amylose percent for CSR-30 was found 23.3% (intermediate amylose content). The percent amylose for the pyramided genotypes varied from 20.7 to 30.2%. All the pyramided genotypes except P–C-4, P–C-19 and P–C-13 showed an ASV of 4 or 6 corresponding to gelatinization temperature (GT). The ASV for CSR-30 was scored 4. All the pyramided BC1F1 genotypes exhibited strong aroma similar to the parents. It was found that the two-gene pyramided BC1F1 genotypes had CSR-30 like characteristics and their grain quality traits were either superior or at par with CSR-30.

Molecular Marker-based Screening for Bacterial Leaf Blight Resistance Genes in Landraces and Cultivars of Rice in Gujarat

Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating disease and limiting factor for rice productivity worldwide. Since BLB pathogens are difficult to manage through chemical and other available cultural practices, development of host plant resistance is the most effective means of disease management. So far survey for resistance donor at molecular level in rice is scanty and the landraces of Gujarat state of India have received little attention. Therefore, a molecular screening was conducted to identify the presence of major BLB resistance genes Xa4, xa5 and Xa21 in landraces and local cultivars of Gujarat. In order to confirm the presence of BLB resistance genes, 25 genotypes were screened using PCR analysis. 10 genotypes (IR20, IR64, IR72, NWGR1095, NWGR2014, IET14726, GR7, GR102, Pankhali 203 and Ratna) were found to be Xa4 positive. In contrast, only IET18483 genotype was found to be Xa21 positive. Interestingly, not a single genotype was found to be positive for xa5 BLB resistance gene. The status of BLB resistance genes in landraces and cultivars of Gujarat will help design BLB-resistance breeding programs.

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC3F1 three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC3F2 generation.

Stability of Four New Sources of Bacterial Leaf Blight Resistance in Thailand Obtained from Indigenous Rice Varieties

Bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases in rice production. Breeding varieties specifically for their resistance to BLB disease is therefore an efficient and cost-effective strategy. However, the resistance gene for BLB can be race and non-race specific, meaning it is often overcome by the pathogen. The identification of new sources of resistance genes for Xoo is crucial in rice breeding programmes. In this study, six rice varieties were assessed using six Xoo isolates in multiple screening conditions. The GGE biplot analysis considers both genotype (G) and genotype environment (GE) interaction effects and demonstrates GE interaction. The first two principal components (PCs) accounted for 95.46% of the total GE variation in the data. Based on lesion length and stability performance, Phaladum was the most ideal genotype against all Xoo isolates in the four screening conditions. The results relayed that Phaladum indigenous rice varieties could be considered as new sources of bacterial leaf blight resistance in Thailand. In the future, the BLB resistance gene in this variety will be identified in regard to mode of inheritance and used as parental line in rice breeding programmes for resistance to BLB.

Marker assisted selection for bacterial leaf blight resistance in segregating populations of Karma Mahsuri

Marker Assisted Selection for Bacterial Leaf Blight resistance in Segregating Populations of Karma Mahsuri Bacterial leaf blight (BLB) of rice is caused by Xanthomonas oryzae pv. oryzae (Xoo), this is a major disease of rice in several countries. The present investigation was undertaken with the objective to develop high yielding rice varieties possessing broad spectrum durable resistance by transferring bacterial leaf blight (BLB) resistant genes viz., xa5 xa13 and Xa21 from a donor IRBB59 and a popular high yielding rice variety i.e., Karma Mahsuri, susceptible to BLB and were selected as parents. The presence of target genes by using gene linked primers viz., xa5R, xa5S, xa13 promoter and Xa21F/R was done. Segregating population was advanced to BC2 F3 generation and foreground selection was done using gene linked markers. Genetic analysis in BC2 F3 populations confirmed the presence of genes (xa5, xa13 & Xa21) governing BLB resistance in 22 lines. p>

Morpho-Molecular Screening for Bacterial Leaf Blight Resistance in Some Rice Lines and Varieties

Bacterial leaf blight (BLB) is one of the major and oldest diseases of rice. The best cost effective management of this disease is the development of resistant cultivars. Morphological and molecular screening are widely used approach in the recent scenario for identifying BLB resistant cultivar. In present study, two experiments were carried out to identify the BLB resistant rice lines and varieties. An active strain of Xanthomonas oryzae pv. oryaze (Xoo.) BXO-09 was used for the inoculation experiments. Then, morphological rating was done to find out the resistant rice lines and varieties whereas three RFLP primers (RG136, RG556 and pTA248) were used for identifying the BLB resistant genes. Through morphological analysis, the rice line RC251 was recorded as susceptible where the rest of the rice lines resulted moderately susceptible or moderately resistant. Among the rice varieties, BR-26, BRRI Dhan31, IRBB5, IRBB21, IRBB60, IRBB65, and Kumragur were recorded as resistant where BR-11, Binadhan-8, and Binadhan-10 were found highly susceptible to BLB. In case of RFLP marker based analysis, a specific type of banding pattern was observed for IRBB5 for RG556 primer which indicated the presence of xa5 gene. For RG136 primer 8 kb single band was formed for different lines and varieties whereas only IRBB60 showed 2 fragments of 8 kb and 5 kb which also indicated the presence of xa13 gene. Finally, DNA amplification of 27 rice lines and varieties using pTA248 primer showed the band at 4 kb position for the BLB resistant lines and varieties and 3.7 kb position for the susceptible lines and varieties. The findings of morpho-molecular characterization information could be useful to the breeders for further planning of developing BLB resistant cultivars.

English

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has now become one of major rice diseases. During recent years, its occurrence has increased in Pakistan, particularly in ‘Kallar tract’ which is well-known for quality rice cultivation. Development of new resistant varieties by utilizing the available resistant gene-pool is most reliable breeding technique for the plant breeders. The present study was conducted to explore the genetic resources regarding presence and absence of BLB resistance genes Xa4 , xa5 and Xa21 through DNA marker technology. Effectiveness of Xa21 followed by xa5 and Xa7 genes has been reported to confer more resistance level for BLB in traditional rice belt of Pakistan. For this purpose, eighty (80) rice genotypes comprising of diverse origin including three isogenic lines viz., IRBB4 (carrying Xa4 ), IRBB5 (carrying xa5 ) and IRBB21 (carrying Xa21 ) as positive resistant gene checks and IR24 (carrying none of these) as negative gene check were genotyped. DNA fingerprinting results indicated the presence of Xa4 gene in 41 entries, while 14 lines were positive for xa5 gene. Only one local line was carrying Xa21 gene along with Xa4 . Thus, the present study will not only be helpful for rice breeders to develop new rice varieties carrying disease resistant genes, but will also provide the donors to incorporate / pyramid disease resistant genes in popular rice varieties like Super Basmati and Basmati 515 which are susceptible to BLB disease. Keywords: Molecular screening, rice germplasm, Xa4, xa5 and Xa21 genes, bacterial leaf blight

Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance

Bacterial Leaf Blight (BLB) is the most important yield limiting factor in Nepalese rice. BLB resistance rice varieties are highly demanding in the country. Breeding efforts for developing disease resistant depends on availability and use of resistant gene donors. Nepalese rice landraces could be the source of resistant gene. Therefore, ninety six Nepalese rice accessions were screened using eight Simple Sequence Repeats (SSR) markers and one Sequence Tagged Sites (STS) marker for presence and absence of BLB resistance gene. We have detected BLB resistance gene Xa-10 on five accessions, Xa-13 on six accessions, Xa-7 on 23 accessions, Xa-3 and Xa-4 on 52 accessions, Xa-5 on 25 accessions, Xa-8 on 30 rice accessions. No any rice accessions tested have Xa-21. Similarly, 17 rice accessions showed three and more than three BLB resistance genes. Presence of Xa-13 on susceptible check variety CNTRL-85033 confirmed that this resistant gene is not working in Nepalese rice field. Therefore, Nepal need to pyramide the BLB resistant genes for durable resistance.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 35-39