STUDIES ON THE BIOLOGY OF POTATO CYST NEMATODES

(GLOBODERA SPP.) UNDER NORDIC CONDITIONS FOR

IMPROVING MANAGEMENT AND REGULATION IN

NORWAY.

Ricardo HolgadoNIBIO, Norwegian Institute of Bioeconomy Research, Norway

STUDIES ON THE BIOLOGY OF POTATO CYST NEMATODES

(GLOBODERA SPP.) UNDER NORDIC CONDITIONS FOR

IMPROVING MANAGEMENT AND REGULATION IN NORWAY.

OBJECTIVES

– Main objective:

To increase the scientific basis for amending the management system for PCN, Globodera spp under Nordic conditions.

– Sub-objectives:

(1) To determine the virulence occurring in selected PCN populations.

(2) To establish the decline rate in PCN numbers and infectivity in selected field soils.

(3) To explore into the occurrence and role of microorganisms antagonistic to PCN.

(4) To evaluate the decline of PCN numbers in field trials with an early potato crop or Solanum sisymbriifolium

(5) To characterize the degree of resistance of selected potato varieties available in Norwegian market

(6) To perform exploratory studies on the PCN- Potato- Pathosystem

PROJECT COLLABORATIVE PARTNER• National

• The Norwegian Food Safety Authority

• The Norwegian Agricultural Extension Service

Nord-Trøndelag, Rogaland, SørØst. Viken Agder, og Potet Forum.

• Potato Industry

(Maarud, AL Gartnerhallen, Norsk Bondelag, Settepotetdyrkernes landslag)

• Farmers

• International

• Julius Kühn-Institut, Germany.

Dr. Björn Niere, Dr. Holger Heuer and Dr. Andreas Westphal

• Rothamsted Research UK.

Dr. Keith Davies

• Universidad Autonoma de Madrid Spain.

Prof. Francisca del Campo, and Soledad Sanz

(1) TO DETERMINE THE VIRULENCE IN SELECTED POPULATIONS

• The work concentrated on second stage juveniles and cysts of selected Globodera-populations, which will be compared to standard populations of G. rostochiensis(i.e. Great Britain Ro1 “Ecosse” and German Ro5 “Harmerz”) and G. pallida (Duddingston Pa1 and Switzerland Pa2/3 “Chavornay”), G. tabacum, G. artemisiae and G. achilleae.

• All standard populations will be reared in special cultivation units kept in a growth room at Bioforsk/NIBIO in Ås.

• Selected populations will be subjected to : Further studies by molecular techniques in collaboration with JKI.

G. rostochiensis

G. pallida

G. Pallida

Population Pathotype /

virulenece

group

determined

Comments

Trøndelag 07 Pa2/3

Jaren 08 Ro1

Lena 1 (2009)

Lena 2 (2009)

Ro1

Minnesund

(2009)

Ro1

Stokke (2009) Ro1

NLR Agder Ro1

NLR Vestfold Ro1

NLR Rogaland Ro1

NLR SørØst Ro1

Stuvik - No multiplication during this study

Molecular analysed Globodera populations

• Norway: 18 populations

• 8 reference populations for pathotypes

• Plant Protection Agency in Hannover: 10 populations fromNiedersachsen (Germany), for which they routinely determinedthe pathotype (eventually mixed populations)

• The DNA was extracted from grinded cysts using a kit (QiagenTissue DNA Extraction Kit).

• Parasitism genes from PCN including two genes (GrCLE1 and GrCLE4), analysis of the gene GrCLE4 showed.

Ro3

Ro5

Minnesund

Rade

Rade

NLR Vestfold

Rygge

Follo C

Stuvik27ANSI(Ro1)

29JASA(Ro1)

21SIOS(Ro2)

27KUAC(Ro2)

26LIPE(Ro2)

29BEWE(Ro3)

Ro1

Ro3

Ro4

Jaren

29BAHO(Ro1)

29TEKI(Ro2)

29LUSA(Ro2)

Ro2

Lena1

Lena2

Stokke09

NLR Rogaland

29TOBI(Ro4)

Gr-CLE-4 gene: analysis of repeats

• No obvious correlation withpathotype

• vap1 belongs to the SCP/TAPS family of genes, which play key roles in host–pathogen interactions and defense mechanisms of diverse eukaryotes

• Gr-vap1 is strongly upregulated in invasive JJ2 in subventral esophageal glands

• Interacts with an extracellular protease of the plant (Rcr3)

• Probably interferes with plant defense responses

Venom Allergen-Like proteins Gr-vap1 parasitism

gene

PCR products of the vap1 parasitism gene

Pa2 Pa3 Ro1 Ro2 Ro3 Ro3 Ro4 Ro5 H. betae

• All analysed strains of Heterodera and Globodera gave the

expected PCR product, except H. avenae

• The PCR products were cloned and sequenced

Wo146-4 Ro2

29BEWE Ro3

21SIOS Ro2

Wo194 6a Stokke09

Wo146-5 Ro3

Wo194 3 Lena1

Wo194 2 Jaren08

29TEKI Ro2

29LUSA Ro2

29REKI Pa3

29TEGI Ro5

29BOSHA Pa3

Wo194 5 Minnesund

Wo194 4 Lena2

29HAMO Ro5

29JASA Ro1

29LIPE Ro2

29BAHO Ro1

29TOBI Ro4

Wo146-3 Ro1

Wo139-5 Ro1

27EGBE2 Pa3

Wo146-7 Ro4

Wo146-8 Ro5

Wo194 7a Rade

21ANSI Ro1

Wo194 1 Trondelag

Wo146-1 Pa2

Wo146-2 Pa3

Neighbour joining tree of vap-1 sequences

• No obvious correlation with

pathotype or geography

• Not all vap1 from G. pallida

cysts cluster together

Similarity of vap-1 gene sequences of Norwegian populations and reference pathotype

populations

Wo146-5 Ro3

Wo194 2 Jaren08

Wo194 3 Lena1

Wo194 5 Minnesund

Wo146-4 Ro2

Wo194 6a Stokke09

Wo146-7 Ro4

Wo146-8 Ro5

Wo139-5 Ro1

Wo146-3 Ro1

Wo194 4 Lena2

Wo194 7a Rade

Wo194 1 Trondelag

Wo146-1 Pa2

Wo146-2 Pa3

P4/3 H schachtii

• It was shown that several variants of the vap-1 gene are present in each population, and that differences in allele frequencies between populations are minor compared to those found for Heterodera schachtii.

• For the population from Råde substantial heterogeneity of vap-1 variety patterns between cysts was found, while populations from other locations seemed to be more genetically homogeneous.

Phylogenetic tree of mitochondrial scmti sequences

from Norwegian and central European PCN.

– Based on the PCR amplification of ITS regions with species-specific primers (Bulman and Marshall, 1997) all populations from Norway were identified as G. rostochiensis, except population Trondelag which was assigned to G. pallida.

– PCR amplification and sequencing of the non-coding scmtmitochondrial region confirmed the species identification, and grouped all Norwegian populations with nearly identical sequences of the most common European populations

(2) To establish the decline rate in PCN numbers and infectivity in selected field soils.

– Population density decline of the nematode over time was studied in the field. In Norway, the earliest incidences of PCN-infections date back to the 1950ties

– To investigate the decline in PCN infectivity in absence of host plants, quarantined fields ranging from 32, 18 and 12 years were selected.

– These fields were infested by G. rostochiensis (Ro3), G. rostochiensis (Ro1) and G. pallida (Pa 2/3) respectively.

– In each soil sample cysts were extracted and baited on a susceptible potato in pots. After 4 months, the soil was analysed for new cysts.

– Viable PCN were found in all soil samples.

– This study, which is the first of its kind on the Nordic area, demonstrated that in the absence of host-plants G. rostochiensis can survive for 32 years, and G. pallida has survived for 12 years so far.

(3) TO STUDY THE OCCURRENCE AND ROLE OF MICROORGANISMS AS ANTAGONISTS OF PCN.

– The following nematophagous fungi were found:

– Pochonia chlamydiosporia,

– Paecilomyces lilacinus (Purpureocillium lilacinum)

– Catenaria spp.

– Arthrobotrys oligospora

– And the bacteria Pasteruria sp.

Photo Siri Abrahamsen

(4) To evaluate the decline of PCN numbers in field trials with an early potato crop or Solanum sisymbriifolium

TRAP CROPS WITH EARLY POTATO

• The experiments showed that infection was reduced when a susceptible potato variety was harvested early, but that the level of infection increase at late harvest.

• Levels of nematode multiplication rate in susceptible potato Ostara was the highest in the entire period.

• Alternating between susceptible and resistant potato cultivars has shown that the infection in the spring does not increase and is partly reduced towards autumn.

• Trap crop with early potatoes harvesting at 80 and 100 days showed that the reproduction rate of PCN populations varies with geographical position. In Østfold, Agder Vestfold and Rogaland the reproduction rates were approximately similar.

• In North Trøndelag, reproduction rate was much reduced compared with the other counties.

EXPERIMENTS WITH SOLANUM SISYMBRIIFOLIUM

– Breeding lines with quicker germination

– Pion Sis 4004 and Sis 6001 Primed and u-primed seeds are provided Plantbreeder Vandijke Semo BV, Marlijn Hellendoorn-Vos

– Vestfold, Østfold, Rogaland, Agder and North Trøndelag have given different results regarding field establishment and weed control. Germination of S. sisymbriifolium takes more than 3 weeks. Weeds developed rapidly in areas with poor germination of S. sisymbriifoliumand manual weeding was necessary.

– Concerning germination a notable difference between the varieties was observed relating to unprimed and primed seeds.

– However manual weeding was necessary in all the plots.

In Norway PCN life cycle takes form 29 to 40 days.

PCN LIFE CYCLE G. ROSTOCHIENSIS AND G. PALLIDA

– The results confirmed that a second generation of PCN J2s were being produced by both nematode species.

– In the middle of Norway in previous our studies have show that PCN populations decrease when susceptible potato was cultivate. This was unclear for us. The continues hatching of jj2 could give a explanation

– However there are a need for further studies, including varieties and other temperature ranges.

15 december 2015

(5) To characterize the degree of resistance of selected potato varieties available in Norwegian market

– According EU PCN Directive 2007/33 / EC on the management of potato cyst nematodes, the degree of resistance must be reported for all commercial potato varieties. The grades vary from one to nine, where nine represents the highest degree of resistance (Anon, 2006)

– The degree of resistance and tolerance was recorded for 26 selected varieties of potato accessible for Norwegian farmers.

Potato

variety

Score

Saturna 9

Liva 9

Arielle 9

Fakse 9

Solist 9

Van Gogh 9

Aksel 9

Oleva 9

Lady Claire 9

Berber 9

Erika 9

Peik 9

Degree of resistant for G. rostochiensis Ro1 - varieties in Norwegian

marked

Bioforsk Konferansen - Hamar 6. februar, 2014

Potetsort Score

Mozart 8

Folva 8

Asterix 8

Rutt 8

Cerisa 8

Potetsort Score

Bruse 3

Beate 2

Troll 2

Desiree 2

Laila 1

Innovator 1

Pimpernell 1

Mandel 6 1

Mandel 1 1

Kerrs Pink 1

(6) TO PERFORM EXPLORATORY STUDIES ON THE PCN-

POTATO- PATHOSYSTEM

– Elicitor treatment b-aminosmørsyre (BABA), benzothiadiazole (BTH).

– Both elicitor BTH and BABA, in susceptible potato plants (cv. Desiré), reduced the infestation of Globodera rostochensis (Ro1) by the induction of the expression of different defense genes.

– However, extensive research are needed in order to reproduce the data in the field and to establish the capacity and durability of the induced resistance.

Conclusions– It is evident that populations of the same pathotype have varying ability to

multiply on the same potato cultivar.

– There are a genetic variability between populations that needs to be explored and paid more attention to in future management strategies.

– PCN life cycle takes about 35 days before forming young females, and about 40 days for the development of new cysts. Successful use of early potatoes as a trap crop, means that the trap crop needs to be lifted no later than 35 days after sowing.

– The studies confirmed that indeed a second generation of PCN J2s were being produced by both nematode species. However there are a need for further studies.

– S. sisymbriifolium is not a practical alternative for managing PCN in Norway.

– We have demonstrated that G. rostochiensis can survive for 32 years in the absence of host plants in Norway. For G. pallida we have so far recorded a survival time of 12 years.

– The tested varieties had different degrees of susceptibility and resistance to yPCNpathotype Ro1. grade nine was recorded in 12 varieties, grade eight in five varieties grade three in one variety, grade two in three varieties and grade one in five varieties.

Acknowledgements

– To The Research Council of Norway, the Foundation for Research Levy on Agricultural Products, the Agricultural Agreement Research Fund, and Norwegian food industries for funding the project 199604.

– To Farmers and Norsk Landbruksrådgiving SørØst, Viken, Nord Trøndelag, Agder, Rogaland contribution in PCN-early potato studies.

– To Vandijke Semo BV, The Netherlands, for providing seeds of Solanum sisymbriifolium.