Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development.

Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.

Recognition of root exudates by seeds of broomrape (Orobanche and Phelipanche) species.

BACKGROUND AND AIMS: The long co-existence of broomrapes and their hosts within the same environment has culminated in a strong adaptation and effective parasitism. As a first step of specialization in the parasitic process, seed receptors of parasitic plant species vary in their ability to recognize compounds released by their hosts. This work aims to investigate potential patterns for the reception requirements needed to activate germination within Orobanche and Phelipanche species. METHODS: Induction of the germination of seeds of nine Orobanche and Pheliphanche species by root exudates of 41 plant species was studied and subjected to biplot multivariate analysis. KEY RESULTS: A high level of specialization in root exudate recognition was found in Orobanche densiflora, O. gracilis and O. hederae, which germinated almost exclusively in contact with root exudates from the plants they infect in nature. At the opposite extreme, Phelipanche aegyptiaca, P. ramosa and O. minor were highly generalist, germinating when in contact with the root exudates of most plant species. Orobanche crenata, O. cumana and O. foetida showed intermediate behaviour. CONCLUSIONS: A universal germination stimulant for all broomrape species has not being identified to date. The synthetic stimulant GR24 is active against most of the weedy broomrape species, but fails with the non-weedy species tested in this study and with the very recent weedy species O. foetida. In addition, germination behaviour of broomrape species depends on the crop plant tested. Weedy broomrapes with a broad host spectrum respond better to the different exudates released by a wide range of crops and wild species than do non-weedy broomrapes, which have a narrow host spectrum and are more restricted to their host range. Root exudates of many plant species were active in stimulating germination of seeds of Orobanche and Phelipanche species for which they are not described as hosts, representing interesting examples of potential trap crops.

First Report of Crenate Broomrape (Orobanche crenata) on White Lupine (Lupinus albus) Growing in Alkaline Soils in Spain and Egypt.

Crenate broomrape (Orobanche crenata Forsk.) is a parasitic weed known to threaten legume crops since antiquity. It is mainly restricted to the Mediterranean Basin, Southern Europe, and the Middle East where it is an important pest in grain and forage legumes and in some apiaceous crops such as carrot and celery (1). White lupines are cultivated in acid soils, which usually are free of O. crenata infestations. However, breeders are attempting to develop white lupine cultivars adapted to alkaline soils (2). We report here findings of O. crenata infection in field trials of this new lupine germplasm in alkaline soils in experimental farms with a known history of faba bean cultivation and heavy infestation of O. crenata in Kafr El-Sheikh, Egypt and Córdoba, Spain in the spring of 2009. Symptoms were typical of O. crenata infection with reduced growth and emergence of typical O. crenata nonbranched spikes close to the lupine plants. Infection was confirmed by digging up the plants to verify the attachment of the broomrape plant to the lupine. O. crenata plants growing on lupines were fully fertile, producing viable seeds. Plant morphology was typical of O. crenata (1). Voucher specimens were deposited at the Herbarium of the Botanic Department of the University of Córdoba. To our knowledge, this is the first report of O. crenata infecting lupine and is relevant because the expected introduction of alkaline-tolerant lupine cultivars will extend its area of cultivation into fields heavily infested with Orobanche. O. crenata is highly polymorphic and could easily adapt to, recognize, and infect this new host. Development of lupine-adapted O. crenata populations should be monitored because it could represent a major constraint on lupine introduction into alkaline soils. References: (1) D. M. Joel et al. Biology and Management of Weedy Root Parasites. Page 267 in: Horticultural Reviews. Vol. 33. John Wiley and Sons, Inc. Hoboken, NJ, 2007. (2) M. Vishnyakova and A. Mikic, White lupin (Lupinus albus L.) landraces and the breeding for tolerance to alkaline soil reaction. Page 142 in: Second GL-TTP Workshop: Integrating Legume Science and Crop Breeding. Novi Sad, Serbia, 2008.

First Report of Crenate Broomrape (Orobanche crenata) on Lentil (Lens culinaris) and Common Vetch (Vicia sativa) in Salamanca Province, Spain.

Broomrapes (Orobanche spp.) are obligate parasites that infect roots of dicotyledonous plants. Orobanche species are particularly important in southern and eastern Europe, the Middle East, and North Africa. O. crenata (crenate broomrape) has been known to threaten legume crops since antiquity. This parasitic weed is mainly restricted to the Mediterranean Basin, southern Europe, and the Middle East and is an important pest in grain and forage legumes as well as in some Apiaceous crops such as carrot and celery (2,3). In Spain, O. crenata is a well-known problem on grain legumes in the south (Andalucía and Extremadura regions) and along the entire east coast north to the border with France. However, it has never been reported as a problem in central Spain. Castilla-León is the major pea-, vetch-, and lentil-producing area of Spain and was believed to be free of O. crenata. However, widespread and heavy infections of O. crenata (as many as 20 broomrapes per m2) were identified in the spring of 2007 on lentil and common vetch growing in several fields in Salamanca Province, between La Vellés, Palencia de Negrilla, and Aldeanueva de Figueroa, covering an area of approximately 80 km2. Infection of lentil and vetch plants was confirmed by digging up the plants to verify the attachment of the broomrape plant to the lentil or vetch roots. Morphology was typical of O. crenata (2), i.e., large erect plants with single, nonbranched spikes that may reach a height of up to 1 m, bearing many flowers of diverse pigmentation from yellow through white to pink and violet. The calyx is 13 to 18 mm with segments free and bidentate. The corolla is 18 to 28 mm, glandular pubescent, the lips often with lilac veins, lips divergent, large, and not ciliate. The anthers are brown, glabrous, or subglabrous. The filaments insert 2 to 3 mm above the base of the corolla and are hairy at the base with glandular hair at the apex. Voucher specimens were deposited at the Herbarium of the Botanic Department of the University of Córdoba. The heavy and widespread level of infection observed in several fields is most likely explained by an inadvertent introduction of O. crenata seed mixed with seed of lentil and vetch. To our knowledge, this is the first report of O. crenata infecting lentil and common vetch in Salamanca Province and is relevant because the area was considered free of the plant. Remarkably, a recent modeling study suggested that O. crenata might become a problem in central and northern Spain (1) since climatic conditions are suitable for its establishment. The spread of this infestation should be monitored because it could represent a major constraint on legume production in this region. References: (1) J. H. Grenz and J. Sauerborn. Agric. Ecosyst. Environ.122:275, 2007. (2) D. M. Joel et al. Biology and Management of Weedy Root Parasites. Page 267 in: Horticultural Reviews. Vol. 33. John Wiley and Sons, Inc. Hoboken, NJ, 2007. (3) D. Rubiales et al. Euphytica 147:187, 2006.