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Summary The soil of grapevine rootstock mother fields was evaluated as a potential inoculum source of black-foot disease pathogens by using four different techniques: fungal isolation from roots of grapevine seedlings used as bait plants; fungal isolation from roots of weeds; multiplex, nested PCR and qPCR. Four Ilyonectria spp., named I. alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis, were isolated from the roots of bait plants grown in a rootstock mother field. “Cylindrocarpon” macrodidymum was also commonly isolated from weeds collected in rootstock mother fields showing a high rate of isolation. The analysis of soils collected from rootstock mother fields with the multiplex, nested PCR as well as with qPCR showed a high rate of detection of I. macrodidyma-complex from soil DNA samples, while the rate of detection of I. liriodendri was markedly lower in the same DNA samples. Then, the contribution of soils of nursery fields as well as commercial vineyards in increasing the infections caused by black-foot pathogens on grapevine cuttings during the rooting phase in nurseries or in new plantations was also investigated. To this aim we used the same techniques described before. Ilyonectria alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis, were isolated from the roots of bait plants grown in nursery fields. In addition, “Cylindrocarpon” macrodidymum was also commonly isolated from weeds collected in nursery fields showing a high rate of isolation. The results obtained with the multiplex, nested PCR as well as with the qPCR showed a high rate of detection of I. macrodidyma-complex from soil DNA samples collected in nursery fields, being the rate of detection of I. liriodendri markedly lower in the same DNA samples. Regarding the soil of commercial vineyards, three Ilyonectria spp., named I. alcacerensis, I. novozelandica and I. torresensis, were isolated from the roots of bait plants grown in pots filled with soils sampled from ten different commercial vineyards. “Cylindrocarpon” macrodidymum was also frequently isolated from weeds collected in several commercial vineyards. It is interesting to note that in all soil types: roostock mother fields, nursery fields and commercial vineyards, species belonging to I. macrodidyma-complex were the most frequently detected. Finally, the effects of temperature, pH and water potential (Ψs) on mycelial growth, sporulation and chlamydospore production of “C.” liriodendri, “C.” macrodidymum and “C.” pauciseptatum were evaluated in order to provide further information on factors affecting growth, reproductive and survival of these pathogens. All isolates were able to ii
Summary grow over a range of temperatures from 5 to 30ºC, with an optimum temperature between 20 to 25ºC, but they did not grow at 35ºC. Active mycelial growth was observed over a range of pHs, from 4 to 8. Regarding the effect of Ψs, in general, mycelial growth was greater on amended media at -0.5, -1.0 or/and -2.0 MPa compared with that obtained on nonamended PDA (-0.3 MPa), and was reduced at Ψs values lower than -2.0 MPa. Most of the “Cylindrocarpon” spp. isolates sporulated at all temperatures, pHs and water potentials tested. In all studied conditions, “C.” liriodendri had the greatest sporulation capacity compared with “C.” macrodidymum and “C.” pauciseptatum. In general, chlamydospore production was not much affected by temperature, pH and Ψs. Chlamydospores were observed in PDA cultures of all isolates at all pH values studied, while some isolates did not produce chlamydospores at 5 and 10ºC or -4.0 and/or -5.0 MPa. Additionally, in this Thesis, pathogens associated with Petri disease of grapevines were also detected on bait plants and weeds. Cadophora luteo-olivacea, Phaeoacremonium aleophilum, Pm. parasiticum and/or Phaeomoniella chlamydospora were isolated from xylem vessels of bait plants grown both in a rootstock mother field and a nursery field as well as from weeds collected in rootstock mother fields, nursery fields and commercial vineyards, confirming soil and weeds as inoculum sources of these pathogens. iii
Page 1: TESIS DOCTORAL ESCUELA TÉCNICA SUP
Page 5: A mis padres A Quique
Page 8 and 9: Agradecimientos A Arianna Rustignol
Page 13 and 14: Resumen Desde principios de los añ
Page 15: Resumen Es importante destacar que
Page 18 and 19: Resum Mitjançant l’ús de quatre
Page 21 and 22: Content Index Figure index………
Page 23 and 24: Figure Index Figure 1.1. A, black d
Page 25: Table Index Table 1.1. Fungal speci
Page 29 and 30: 1.- GENERAL INTRODUCTION 1.1.- Youn
Page 31 and 32: General introduction associated wit
Page 33 and 34: General introduction perennial host
Page 35 and 36: General introduction chlorosis and
Page 37 and 38: General introduction the species an
Page 39 and 40: General introduction 2006b). In add
Page 41 and 42: General introduction Portugal (Cabr
Page 43 and 44: General introduction produced from
Page 45 and 46: General introduction They are gener
Page 47 and 48: General introduction covering graft
Page 49 and 50: General introduction foot pathogens
Page 51 and 52: General introduction a semi-commerc
Page 53 and 54: General introduction obtained by Bl
Page 55 and 56: General introduction Aroca, A., Gar
Page 57 and 58: General introduction Chaverri, P.,
Page 59 and 60: General introduction Gramaje, D., A
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General introduction Larignon, P. 1
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General introduction Petit, E. and
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General introduction Serra, S., Man
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Chapter 2
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Chapter 2 5. Study the effects of t
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Chapter 3 Detection of black-foot d
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Chapter 3 grapevines as symptomatic
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Chapter 3 been recently used to det
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Chapter 3 of denaturation at 94°C
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Chapter 3 cut grafting machines and
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Chapter 3 macrodidyma-complex in bo
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Chapter 3 obtained by Halleen et al
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Chapter 3 Alaniz, S., Abad-Campos,
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Chapter 3 canes from trunks of infe
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Chapter 3 control with combined hot
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Chapter 4 Detection of black-foot a
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Chapter 4 External symptoms of this
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Chapter 4 Greenhouse experiments So
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Chapter 4 The identification of Ily
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Chapter 4 Table 4.1. Incidence and
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Chapter 4 isolation ranged from 1.0
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Chapter 4 al., 2006), the positive
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Chapter 4 Aroca A, Raposo R (2007)
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Chapter 4 Gramaje D, Mostert L, Arm
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Chapter 4 Whiteman SA, Jaspers MV,
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Chapter 5 Detection and quantificat
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Chapter 5 Based on current data, bl
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Chapter 5 Materials and methods Fie
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Chapter 5 Sensitivity of the qPCR a
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Chapter 5 Table 5.2. Fungal identif
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Chapter 5 In general, qPCR analyses
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Chapter 5 the qPCR techniques have
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Chapter 5 Filion M, St-Arnaud M, Ja
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Chapter 6
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Abstract Weeds as potential hosts W
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Weeds as potential hosts undetected
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A C E F Weeds as potential hosts Fi
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Weeds as potential hosts the E.Z.N.
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Weeds as potential hosts Table 6.1.
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Weeds as potential hosts very varia
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Weeds as potential hosts 256 (mean
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Weeds as potential hosts in the C.
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Weeds as potential hosts 10. Black,
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Weeds as potential hosts disease in
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Weeds as potential hosts 54. Wisler
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Chapter 7 Effects of temperature, p
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Chapter 7 discoloration and necrosi
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Chapter 7 Table 7.1. Sources of iso
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Chapter 7 presence/absence of chlam
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Chapter 7 isolate Cy59 (C. lirioden
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Radial growth (cm day -1 ) Chapter
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Radial growth (as % of control) Cha
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Chapter 7 nonamended PDA (-0.3 MPa)
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Chapter 7 liriodendri isolates and
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Chapter 7 dsRNA on growth rate and
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Chapter 7 Martin M.T., L. Martin, M
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Chapter 8
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Chapter 8 The main objective of thi
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Chapter 8 be investigated is the ro
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Chapter 8 difficult isolation of th
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Chapter 8 isolates were able to gro
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Chapter 8 2010a). Thus, an integrat
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Chapter 8 Dubrowski, S. and Fabriti
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Chapter 8 Pascoe, I. G., and Cottra
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CONCLUSIONS Chapter 9 1.- Inoculum
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