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Actinidia seed-borne latent virus (ASbLV, Betaflexiviridae), was detected at high frequency in healthy seedlings grown from lines of imported seed in a New Zealand post-entry quarantine facility. To better understand how to manage this virus in a dioecious crop species, we developed a rapid molecular protocol to detect infected progeny and to identify a reliable plant tissue appropriate to detect transmission rates from paternal and maternal parents under quarantine environment.The frequency of ASbLV detection from true infection of F1 progeny was distinguished by testing whole seeds and progeny seedling tissues from a controlled cross between two unrelated parents; an ASbLV-infected staminate (male) plant and an uninfected pistillate (female) plant, and the process was repeated with an ASbLV uninfected staminate (male) plant and an infected pistillate (female) plant. Individual whole seeds, or single cotyledons from newly-emerged seedlings, true leaf or a root from those positive-tested seedlings, were assessed for presence of ASbLV by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The virus was detected at a high incidence (98%) in individual seeds, but at a much lower incidence in seedling cotyledons (62%). Since detection results were consistent (P=95%) across the three seedling tissues (i.e. cotyledons, leaves and roots) only cotyledons were tested thereafter to determine ASbLV transmission to F1 progeny. F1 seedlings from three crosses were used to compare transmission rates from infected staminate versus infected pistillate parents. One cross from a single flower used an uninfected pistillate vine pollinated by an infected staminate vine, and two crosses (also from a single flower) used an infected pistillate vine (a sibling of the infected staminate vine), pollinated by either of two unrelated uninfected staminate vines.Cotyledon testing of seedlings from each cross confirmed staminate transmission at high frequency (∼60%), and pistillate transmission at even higher frequency (81% and 86%, respectively).The results show ASbLV is transmitted at very high rates, whether from infected ovules or pollen. Transmission to seedlings is lower than detection in whole seeds perhaps due to ASbLV being sometimes residing on (or within) the seed coat only. The results also show RT-PCR of cotyledons allows non-destructive detection of ASbLV in very young seedlings, and could be used to screen kiwifruit plants in a nursery to avoid virus spread to orchards. Likewise, bulk testing of seed lots can quickly detect infected parent plants (fruit bearing female or male pollinator) already in an orchard.ImportanceActinidia seed-borne latent virus (ASbLV, Betaflexiviridae), was detected at high frequency in healthy seedlings grown from lines of imported seed in a New Zealand post-entry quarantine facility. However there are several technical barriers to detecting the presence of seed transmitted viruses and understanding their biology, which has significance for detection in quarantine and subsequent management under germplasm collections. To overcome this, we developed a rapid molecular protocol to detect infected progeny and to identify a reliable plant tissue appropriate to detect transmission rates from paternal and maternal parents under quarantine environment. Individual whole seeds, or single cotyledons from newly-emerged seedlings, true leaf or a root from those positive-tested seedlings, were assessed for presence of ASbLV by reverse transcription-polymerase chain reaction (RT-PCR) analysis. This was done with seed lots obtained from four separate controlled crosses between ASbLV-infected and ASbLV-uninfected Actinidia chinensis var. deliciosa parents.

The pathogenicity of Alternaria spp. on Malus × domestica in New Zealand has not been investigated fully. Alternaria spp. were isolated from necrotic spots on leaves and fruit rots from various apple cultivars. Pathogenicity of 31 Alternaria spp. isolates was tested on excised ‘Royal Gala’, ‘Golden Delicious’ and ‘Scifresh’ leaves (wounded and unwounded) by measuring lesion diameters. Viability of Alternaria spp. grown from infected leaf material was assessed to compare various storage conditions. Alternaria spp. were consistently isolated from the central brown area of leaf lesions but could not be isolated from outside the leading edge or from the purple region surrounding the lesion. All isolates produced lesions on wounded leaves. Most isolates showed some pathogenicity on at least one cultivar, with lesions on unwounded leaves. Lesions on leaf material caused by Alternaria spp. can be stored for at least 11 weeks by freeze-drying or air-drying leaf material and storing at 0.5˚C or room temperature. Some New Zealand Alternaria spp. isolates on Malus × domestica are pathogenic on fruit and leaves. Further studies are needed to better understand the biology of this pathogen in New Zealand and determine a suitable control programme.

Background Although the most common path of infection for fire blight, a severe bacterial disease on apple, is via host plant flowers, quantitative trait loci (QTLs) for fire blight resistance to date have exclusively been mapped following shoot inoculation. It is not known whether the same mechanism underlies flower and shoot resistance. Results We report the detection of a fire blight resistance QTL following independent artificial inoculation of flowers and shoots on two F1 segregating populations derived from crossing resistant Malus ×robusta 5 (Mr5) with susceptible ‘Idared’ and ‘Royal Gala’ in experimental orchards in Germany and New Zealand, respectively. QTL mapping of phenotypic datasets from artificial flower inoculation of the ‘Idared’ × Mr5 population with Erwinia amylovora over several years, and of the ‘Royal Gala’ × Mr5 population in a single year, revealed a single major QTL controlling floral fire blight resistance on linkage group 3 (LG3) of Mr5. This QTL corresponds to the QTL on LG3 reported previously for the ‘Idared’ × Mr5 and an ‘M9’ × Mr5 population following shoot inoculation in the glasshouse. Interval mapping of phenotypic data from shoot inoculations of subsets from both flower resistance populations re-confirmed that the resistance QTL is in the same position on LG3 of Mr5 as that for flower inoculation. These results provide strong evidence that fire blight resistance in Mr5 is controlled by a major QTL on LG3, independently of the mode of infection, rootstock and environment. Conclusions This study demonstrates for the first time that resistance to fire blight caused by Erwinia amylovora is independent of the mode of inoculation at least in Malus ×robusta 5.

Experiments were carried out to compare fire blight resistance screening methodologies used in the German and New Zealand pipfruit breeding programmes. The small differences between the respective screening methodologies did not result in significant differences in the incidence of fire blight infection or the amount of resistance observed in plants. Differences in bacterial growth media, inoculum suspension and inoculation technique did not have a significant effect on the incidence of fire blight infection or the extent of resistance expression in various apple cultivars. Some of the Erwinia amylovora isolates had a significant effect on the level of disease expression on different cultivars. These differences can be used as tools to identify resistance quantitative trait loci in different cultivars. Results from these experiments have led to standardisation of screening methods between the two countries and provided confidence in comparing the findings of the New Zealand/German collaborative resistance research programme.

Several Actinidia spp. genotypes exhibit crown gall-like symptoms in both roots and canes. Galls form on roots and pruning wounds of canes. Investigations were undertaken to determine if an Agrobacterium species was responsible for gall formation in the Actinidia spp. material. Macerated galls were plated onto King’s B and various selective Agrobacterium agars, e.g. 1A and Roy & Sasser media. Bacterial isolates were tested by PCR for the presence of tumour-inducing (Ti) plasmids. Isolates that tested positive for the Ti plasmid were subsequently tested for pathogenicity by inoculation onto cut carrot discs, Nicotiana glutinosa and Solanum lycopersicum, and assessed for gall formation. Bacterial isolates that tested positive by PCR for the Ti plasmid were an orange tan colour on selective medium 1A, and clear with a red centre on the Roy & Sasser medium. Galls formed on cut carrots, S. lycopersicum and N. glutinosa after inoculation of Ti-positive bacterial isolates. Initial results indicate that an Agrobacterium species is associated with the formation of galls in Actinidia seedlings. However, biochemical characterisation and confirmation of Koch’s postulates using Actinidia species are needed for verification of this result.

Fire blight a bacterial disease caused by Erwinia amylovora is an ongoing problem for pipfruit growers with few control options available Most commercial cultivars and rootstocks are highly susceptible to the disease Breeding of fire blightresistant scions and rootstocks to manage the disease is a major goal of the New Zealand apple breeding programme The main mode of disease establishment is through flowers However the breeding programme currently evaluates disease resistance through shoot inoculations This study compared the degree of resistance in 109 progeny from a Royal Gala times; Malus robusta Robusta 5 family assessed by shoot inoculation and by floral inoculations Results indicate that the two measures of resistance do not correlate well and that different quantitative trait loci may be involved in flower and shoot resistance Management of fire blight through the implementation of resistant cultivars will require resistance screening on both shoot and flower assessments

Herbaceous indicator bioassays are used to screen for unwanted plant viruses on imported plant material To optimise virus detection the effect of plant nutrition was investigated to determine whether this plays a role in virus symptom expression and severity Apple stem grooving virus and Tobacco ringspot virus were inoculated onto a range of herbaceous indicator species grown in potting mix supplemented with differing rates of slowrelease fertiliser Higher rates of fertiliser resulted in better plant nutrition and a greater incidence of virus expression and more severe expression of disease in the seven indicator plant species Overall disease assessments indicated that plant nutrition affects plant growth virus infection rate and virus symptom expression As virus infection affects plant growth and leaf colour good plant nutrition is important to avoid masking virus symptoms and to optimise the detection of viruses in postentry quarantine facilities

Pollen is traded internationally as a source of germplasm and for pollination. Thirty-nine viruses and five viroids are known to be pollen transmitted. We investigated whether reverse transcription-polymerase chain reaction (RT-PCR) could be used to detect viruses reliably in pollen. Four extraction methods yielded nucleic acid in appropriate quantity and quality from Tobacco ringspot virus (TRSV)-infected pollen for RT-PCR

Fire blight, caused by Erwinia amylovora (Ea), is a destructive disease of Malus (apple), Pyrus (pear) and some woody ornamentals in the rose family (Rosaceae). The goal of this project is to use a functional genomics approach to develop tools to breed fire blight resistant apples. Suppression subtractive hybridization (SSH) and cDNA-AFLP analysis were used to identify 650 expressed sequence tags (ESTs) associated with fire blight from Ea-challenged apple leaf tissue. ESTs were ranked for their potential impact on resistance based on bioinformatics and inferences drawn from model systems. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers derived from highly ranked fire blight-associated ESTs were mapped in a ‘M.9’ × ‘Robusta 5’ population in which a major QTL for fire blight resistance has been located on Linkage Group 03. Highly ranked fire blight-associated ESTs were mapped to this QTL, as well as to the positions corresponding to the location of at least two QTLs reported in other populations. A secretory class III peroxidase mapped to the LG03 fire blight resistance QTL and a serine/threonine protein kinase mapped at a 4 cM distance. To date, 6 out of 28 candidate fire blight resistance gene markers that have been mapped, have co-located to or near known fire blight resistance QTLs. This research will facilitate new methods of marker-assisted selection to efficiently breed superior apple cultivars with fire blight resistance.

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R 2 = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R 2 = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.

Ascospore production from pseudothecia in apple leaf litter in the spring is a critical step in the disease cycle of Venturia inaequalis Leaf litter management is potentially an important component of an integrated pest and disease management strategy An orchard study was carried out in Hawkes Bay to investigate the relationships between the incidence of black spot on leaves in autumn the density of apple leaf litter on the orchard floor in spring and the number of ascospores produced in spring A total of 22 blocks on eight orchards were surveyed for black spot incidence in autumn 2000 The following spring remaining leaf litter was measured and ascospore production was quantified using spore traps Autumn black spot levels and spring litter levels were accurate predictors of spring ascospore production Management strategies employed on different blocks influenced spring leaf litter and ascospore levels Blocks with high autumn black spot generally had high spring ascospore production Management practices that substantially reduced litter levels resulted in reduced V inaequalis inoculum

Helicobasidium purpureum (anamorph Rhizoctonia crocorum) is the causal organism of the carrot disease violet root rot, common in the Ohakune region of New Zealand. Helicobasidium purpureum has proven a difficult organism to isolate and grow in culture, confounding diagnosis of early infections. To enable early diagnosis of the disease, a conventional PCR assay was developed with the primer sequences (HelicoPurp1 and HelicoPurp2, 101bp amplicon) targeting part of the internal transcribed spacer (ITS) region of the organism. The assay detected all 15 H. purpureum isolates collected and maintained by our laboratory in pure culture. The assay was also robust enough to detect the fungi’s full range of inoculum types from the field (sclerotia, hyphae, mycelial mats and ‘black sheaths’), and was also able to detect the fungus when it was in association with plant tissues. Samples required washing, as soil inhibited this assay.

Violet root rot (VRR), caused by the fungus Helicobasidium purpureum (anamorph Rhizoctonia crocorum), is a common problem for carrot growers in the Ohakune region of New Zealand. VRR can still be problematic even after many years of fallow, suggesting that alternative hosts may be maintaining and harbouring the disease. Seeds from a selection of common weed and pasture (ryegrass, clover and plantain) species sourced from the Ohakune area were planted into pots of steam-sterilised Ohakune soil and grown in the glasshouse. Following seedling establishment, pots were inoculated with H. purpureum. Four and a half months post inoculation, all plants were assessed visually, and then well-rinsed root samples were taken for DNA extraction and PCR testing. Of the nine weed species assessed, all tested positive for H. purpureum by PCR, and seven had visual symptoms on plants. For the twelve pasture species assessed, ten tested positive for H. purpureum by PCR, while eight had visual symptoms on plants. Some plants, in particular various clovers, plantain and dock, had numerous sclerotia on the outside of roots. These results indicate that alternate hosts in pasture or fallowed fields are capable of harbouring H. purpureum.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Background: Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus 'Robusta 5'. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results: When several defined E. amylovora strains were used to inoculate three progenies from international breeding programs, all with 'Robusta 5' as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand 'Malling 9' X 'Robusta 5' population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein (MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German 'Idared' X 'Robusta 5' population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene (HSP90). In the US 'Otawa3' X 'Robusta5' population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. Conclusions: The results suggest that the upper region of 'Robusta5' linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

The recent 8th Australasian plant virology workshop in Rotorua, New Zealand, discussed the development of a New Zealand database of plant virus and virus-like organisms. Key points of discussion included: (i) the purpose of such a database; (ii) who would benefit from the information in a database; (iii) the scope of a database and its associated collections; (iv) database information and format; and (v) potential funding of such a database. From the workshop and further research, we conclude that the preservation and verification of specimens within the collections and the development of a New Zealand database of plant virus and virus-like organisms is essential. Such a collection will help to fulfil statutory requirements in New Zealand and assist in fulfilling international obligations under the International Plant Protection Convention. Sustaining such a database will assist New Zealand virologists and statutory bodies to undertake scientifically sound research. Establishing reliable records and an interactive database will help to ensure accurate and timely diagnoses of diseases caused by plant viruses and virus-like organisms. Detection of new incursions and their diagnosis will be further enhanced by the use of such reference collections and their associated database. Connecting and associating this information to similar overseas databases would assist international collaborations and allow access to the latest taxonomic and diagnostic resources. Associated scientists working in the areas of plant breeding, export phytosanitary assurance and in the area of the conservation estate would also benefit from access to verified specimens of plant viruses and virus-like organisms. We conclude that funding of a New Zealand database of virus and virus-like organisms and its associated collections should be based partly on Crown funds, as it is a nationally significant biological resource.

A sensitive conventional RTPCR assay has been developed for an uncharacterised virus within the family Betaflexiviridae prompted by detection of the virus in Actinidia seedlings held in postentry quarantine that had been grown from openpollinated seed Infection is asymptomatic and presently assessed as lowtozero biosecurity risk The assay targets the RNAdependent RNA polymerase (RdRP) of the virus primer sequences (BetaF2 and BetaR2 278bp amplicon) being based on a partial RdRP sequence for the virus The assay detects the nine virus isolates found to date in a range of sample types (fresh leaf stem cambium archived RNA) The assay which includes a multiplexed internal control has been accredited by International Accreditation New Zealand for Actinidia testing To support management of the virus the assay and associated RNA extraction method have been applied to testing dried Actinidia seed directly They have been found sufficiently sensitive to detect the virus in individual seeds Initial results show efficient transmission of the virus from infected pollen to the seed

Current tools for the control of fire blight disease of apples caused by Erwinia amylovora have limitations, including the increasing pressure by markets against the use of streptomycin. Coast Manuka and Coast Lema ® Oil products have been previously shown to provide control against some bacterial, fungal and yeast diseases. Experiments were carried out to determine whether either of these products provided control against Erwinia amylovora. Coast Lema Oil (0.5, 1, 2, 3, 4% w/v) inhibited E. amylovora when added to a bacterial suspension. Coast Manuka Oil (0-4% w/v) failed to inhibit E. amylovora when added to the bacterial suspension. It was also demonstrated that Coast Lema Oil (0.5, 1, 2, 3, 4% w/v) and Coast Manuka Oil (0.5, 1, 2, 3, 4% w/v) inhibited E. amylovora replication when added to agar. Filter paper discs soaked in Coast Lema Oil (2, 3, 4% w/v) caused small inhibition zones around the product when placed directly onto E. amylovora. Coast Manuka Oil (0-4% w/v) was unsuccessful in causing inhibition zones around the discs when placed directly onto E. amylovora. These initial results indicate that Lema oil has the potential to control fire blight in pipfruit trees.

Surveys to determine the phytosanitary status of apple (Malus) and pear (Pyrus) trees in New Zealand were conducted from 2005 to 2006 A total of 188 symptomatic and nonsymptomatic trees from various geographical locations were tested for the presence of a number of viruses and viroids by RTPCR All Malus samples were tested for Apple chlorotic leaf spot virus (ACLSV) Apple stem grooving virus (ASGV) Apple stem pitting virus (ASPV) Apple mosaic virus (ApMV) Apple scar skin viroid (ASSVd) Apple dimple fruit viroid (ADFVd) Cherry rasp leaf virus (CRLV) and Tomato ringspot virus (ToRSV) ACLSV was detected in 48 ASGV in 36 ASPV in 61 and ApMV in 45 of samples tested ASSVd ADFVd CRLV ToRSV were not detected in any of the 165 sampled Malus plants which provides evidence that they are not present in New Zealand All Pyrus samples were tested for ACLSV ASGV ASPV ApMV Pear latent virus (PeLV) and Pear blister canker viroid (PBCVd) ACLSV was detected in 17 ASGV in 13 ASPV in 17 and ApMV in 4 of sampled Pyrus trees PeLV and PBCVd were not detected in any of the 23 sampled symptomatic Pyrus trees which provides evidence that PeLV and PBCVd are not present in New Zealand

In 2012, trials were established in four kauri forest sites severely affected by kauri dieback (Phytophthora agathidicida = P. taxon Agathis, PTA) to determine the potential of phosphorous acid (phosphite) as a control tool. Baseline assessments of 162 trial trees included canopy disease rating, trunk lesion dimensions and lesion activity (recent bleeding/ oozing). Phosphite (Agrifos®600) at concentrations of 7.5% or 20% was injected (20 ml) at 20-cm intervals around the trunk. Control trees were left untreated. After 1 year, half the previously injected trees were re-injected, in all cases with 7.5% phosphite. Phytotoxicity symptoms (leaf yellowing, browning or leaf/twig abscission) were noted in some phosphite- injected trees, particularly where the 20% concentration was used. After 3 years, many more trunk lesions remained active (expressing ooze, continued expansion) in untreated trees (58.5%) than in phosphite-treated trees (0.8%). Average lesion expansion after 3 years was 12.7 cm in untreated and 0.4 cm in phosphite-treated trees.