First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi.

On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

First Report of Rice yellow mottle virus on Rice in Burundi.

Since the mid-1980s, rice cultivation has expanded rapidly in Burundi to reach approximately 50,000 ha in 2011. In 2007, leaf mottling, reduced tillering, and stunting symptoms were observed on rice at Gatumba near Bujumbura, causing small patches in less than 10% of the fields. Rice yellow mottle virus (RYMV, genus Sobemovirus), which has seriously threatened rice cultivation in Africa (1) and was recently described in the neighboring Rwanda (3), was suspected to be involved because of similar symptoms. To identify the pathogen that caused the disease in Burundi, a survey was performed in the major rice-producing regions of Burundi and Rwanda. Six locations in Burundi and four in Rwanda were investigated in April and October 2011. Disease incidence in the fields was estimated to be 15 ± 5%. Symptomatic leaves of 24 cultivated rice plants were collected and tested by double antibody sandwich-ELISA with polyclonal antibodies raised against the RYMV isolate Mg1 (2). All tested samples reacted positively. Four isolates were inoculated on susceptible Oryza sativa cultivar IR64 (2). The typical symptoms of RYMV were reproduced 7 days after inoculation, whereas the noninoculated controls remained healthy. Total RNA was extracted by the RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) from 12 samples. The RYMV coat protein gene was amplified by RT-PCR with primers 5'CGCTCAACATCCTTTTCAGGGTAG3' and 5'CAAAGATGGCCAGGAA3' (3). The sequences were deposited in GenBank (Accession Nos. HE654712 to HE654723). To characterize the isolates, the sequences of the tested samples were compared in a phylogenic tree including a set of 45 sequences of isolates from Rwanda, Uganda, western Kenya, and northern Tanzania (2,3). Six isolates from western Burundi, namely Bu1, Bu2, Bu4, Bu7, Bu10, and Bu13 (Accession Nos. HE654712 to HE654716 and HE654718), and the isolate Rw208 (HE654720) from southwestern Rwanda, belonged to strain S4-lm previously reported near Lakes Malawi and Tanganyika. They fell within the group gathering isolates from the western Bugarama plain of Rwanda (3). The isolates Bu16 (HE654719) and Bu17 (HE654717) from Mishiha in eastern Burundi belonged to strain S4-lv previously reported around Lake Victoria. However, they did not cluster with isolates from the eastern and southern provinces of Rwanda. They were genetically more closely related to isolates of strain S4-lv from northern Tanzania. Overall, the phylogeography of RYMV in Burundi and Rwanda region was similar. In the western plain of the two countries, the isolates belonged to the S4-lm lineage, whereas at the east of the two countries at midland altitude, they belonged to the S4-lv lineage. The presence of RYMV in Burundi should be considered in the future integrative pest management strategies for rice cultivation in the country. References: (1) D. Fargette et al. Annu. Rev. Phytopathol. 44:235, 2006. (2) Z. L. Kanyeka et al. Afr. Crop Sci. J. 15:201, 2007. (3) I. Ndikumana et al. New Dis. Rep. 23:18, 2011.

Accumulation of tocopherols and tocotrienols during seed development of grape (Vitis vinifera L. cv. Albert Lavallée).

Tocopherols and tocotrienols are present in mature seeds. Yet, little is known about the physiological role and the metabolism of these compounds during seed development. Here we present data on tocopherol and tocotrienol accumulation during seed development in Vitis vinifera L. cv. Albert Lavallée (Royal). This species was chosen for its ability to synthesize both tocopherols and tocotrienols. It is shown here for the first time that during seed development there are significant differences in localization and accumulation kinetics of tocopherols and tocotrienols. Tocopherols are found homogeneously dispersed throughout all tissues of the seed, in concentrations ranging from 20 to 100 microg tocopherol per g dry weight. Tocopherol levels decrease gradually during seed development. In contrast, tocotrienols are only found in the endosperm of the seeds, accumulating in a sigmoid fashion during the maturation period of seed development. Tocotrienol levels were found to be (54+/-7.4) microg/g dry seed in 90-day-old seeds of V. vinifera L. Furthermore, tocotrienol biosynthesis is demonstrated in these seeds during tocotrienol accumulation and in an endosperm fraction isolated at 75 days after flowering.

Genetic analysis of resistance to blast in the Vietnamese rice cultivar 'Chiembac'.

The resistance to rice blast disease in the Vietnamese traditional rice cultivar 'Chiembac' was studied. The blast resistance spectrum in 'Chiembac' and 15 rice differentials carrying different known resistance genes was identified using 25 Pyricularia grisea isolates derived from 15 AFLP lineages from the North, Center and South of Vietnam. None of the differential lines carrying a single resistance gene could effectively control all tested Vietnamese blast isolates. 'Chiembac' showed a different resistance pattern compared to that of the differential lines. A cross between 'Chiembac' and 'CR203', an improved rice cultivar, was made and the F2 population was used for characterization and mapping of the resistance genes in 'Chiembac'. Genetic analysis showed that the resistance against two representative isolates from two predominant lineages, VT7 and 12, in 'Chiembac' was controlled by the single dominant genes Pi-VT7 and Pi-I2. The resistance gene Pi-VT7 was closely linked to Pi-I2 and was mapped to chromosome 12 using the framework mapping population 'IR64' x 'Azucena' of 124 double haploid progenies. The resistance to the Vietnamese blast isolate VT7 in 'IR64' was also studied. The latter was controlled by one locus with major effect located on chromosome 12 and mapped closely to the AFLP marker NIN080, which was also tightly linked to the resistance gene Pi-VT7 in 'Chiembac'. Thus, the resistance locus Pi-VT7 and the resistance locus in 'IR64' probably belong to a cluster of resistance genes.

Bean Anthracnose: Virulence of Colletotrichum lindemuthianum Isolates from Burundi, Central Africa.

The diversity of Colletotrichum lindemuthianum is a major limiting factor in control of anthracnose on bean (Phaseolus vulgaris), and race characterization of this pathogen is an important tool in breeding programs. Race characterization has been carried out on isolates from North, Central, and South America; Europe; and Asia, but little or no information exists on the diversity of C. lindemuthianum in Africa. In this work, 12 isolates from the major bean-growing areas of Burundi, Central Africa, were characterized. Their virulence was tested on 12 bean differential cultivars (1) and on 4 bean cultivars commonly grown in Burundi: 2 from local germ plasm (Muyinga-1 and Urubonobono) and 2 from Colombia (A 321 and Calima). Detached unifoliate bean leaves from 8-day-old plants were placed on a humid surface in trays and sprayed until runoff with a suspension of 106 spores ml-1. Trays covered with transparent plastic sheets to keep a minimum relative humidity of 92% were incubated at 20°C. Seven days after inoculation, symptoms were evaluated for severity on a scale of 1 to 9. Leaves scored as 1 to 3 were considered resistant. Races were characterized according to a numerical binary system (1). Nine races were identified: 9, 69, 87, 384, 385, 401, 448, 449, and 485. Seven of these races (9, 69, 87, 384, 401, 448, and 485) were described for the first time in Africa. Races 401 and 485 have not yet been reported in the literature. The most susceptible differential cultivars were Michelite, PI 207262, To, and Mexico 222. Muyinga-1, Urubonobono, and A 321 were sensitive to nine, six, and five races, respectively. There is a high diversity of C. lindemuthianum in Burundi, and the local germ plasm tested is very susceptible to the characterized races. Breeding programs in Burundi should focus on lines and cultivars, such as Tu, AB 136, G 2333, and Calima, that are resistant to all the races characterized in this study. Reference: (1) M. A. Pastor-Corrales. Phytopathology 81:694, 1991.