Neonectria ditissima is the fungal cause of costly European Canker (EC) in apple trees. A range of secondary metabolites were found at higher concentrations in infected twigs than in disease free twigs. Apple trees were then experimentally inoculated with N. ditissima and analyzed periodically until EC symptoms were visible at 12-13 weeks post-inoculation. Established destructive detection methods used were microscopy, which showed extensive hyphal penetration by 8 weeks post inoculation, and qPCR analyses, which confirmed the presence of N. ditissima. Headspace solid-phase microextraction GC-MS data showed significantly higher concentrations of styrene in apple twigs at six weeks after inoculation, and LC-MS data showed phloretin, triterpene acids, and 1-benzoyl β-d-glucose at raised concentrations after this time. Therefore, these compounds could be useful indicators of N. ditissima infection prior to visible canker formation, suitable for nondestructive disease detection development after further research on apple variety, pathogen specificity, and on field detection technology.

Bull’s eye rot, caused by Phlyctema vagabunda, is an important postharvest rot of apples. A combination of laboratory and field trials were conducted to examine the relationship between infection and status of apple lenticels. Fruit were hydrated and then inoculated, and climatic factors were compared with inoculations and natural infections in the field. In laboratory trials it was shown that increased lenticel density and size led to more infections. Hydration distended lenticel diameter, and dehydration contracted lenticels. A strong relationship with wind run, leaf wetness and temperature were shown to be important for laboratory inoculations of monthly harvested fruit, natural infections in trap fruit experiments, and monthly field inoculations. It was hypothesised that high wind run (> c. 240 km/day) reduced fruit susceptibility by promoting lenticel closure, and when lenticels were open, temperature limited the formation of an infection stroma. If the stroma did not form, postharvest expression of disease was negligible. Leaf wetness over 28 days was also an important factor for infection, probably because of the effect on micro-crack formation and resealing of lenticels during maturation by components of the cuticle. From these results, pre-harvest application of compounds that close lenticels could provide some control of bull’s eye rot without the need for fungicides.

Chapter 17 covers diseases of kiwifruit vines and fruit. Most of the chapter focuses on fungal pathogens although viral diseases are also mentioned. The distribution, symptoms, disease cycle and control measures are described for each pathogen. Vine diseases are divided into those affecting the root, crown, trunk and canopy, with specific mention of kiwifruit vine decline syndrome. Postharvest diseases of fruit are divided into ripe rots or wound rots, with botrytis rot discussed as a specific example of the latter.

Bull’s eye rot is a postharvest storage disease of apples. Until now the cause of this disease in New Zealand was not clear. A survey of 6880 apples from five regions of New Zealand over two seasons was conducted. Neofabraea malicorticis and N. perennans were not found. One hundred and seventy-nine isolates were identified as Phlyctema vagabunda by specific polymerase chain reactions and/or sequencing the β-tubulin gene region followed by phylogenetic analysis. Two isolates were identified as N. kienholzii . Previous records of the presence of N. malicorticis and N. perennans in New Zealand were based on spore morphology and presence in pruning wound cankers. There is overlap in spore morphology for N. malicorticis , N. perennans and P. vagabunda , accounting for misidentifications. Based on our results it is likely that previous records were P. vagabunda , which can also infect pruning wounds.

Neonectria ditissima causes a debilitating apple tree canker disease. We determined the efficacy of polymerase chain reaction primers, originally designed for European strains, by sequencing New Zealand strains. The concatenated ribosomal inter-transcribed spacer and β-tubulin gene regions of 17 New Zealand isolates were compared with those of two European strains by phylogenetic analysis. New Zealand and European isolates of N. ditissima were in the same clade, suggesting that there has been little change in these gene regions following introduction to New Zealand. There was 100% homology with Bt-FW135 and Bt-RW284 primers. Based on sequencing 17 New Zealand isolates from several locations, these polymerase chain reaction primers can be relied upon to amplify New Zealand isolates of N. ditissima.

'Hass' avocado fruit were wound-inoculated in the orchard at monthly intervals for 7 months with labelled strains of Colletotrichum acutatum and Phomopsis sp. The strains were single-spore isolates that were selected for their lack of ability to metabolise nitrate (nit strains). Fruit were harvested a month after the last inoculation and placed in the coolstore for 28 days, then placed at 20 degrees C and assessed when ripe. Rot lesions were aseptically removed and placed on potato dextrose agar. Any resultant C. acutatum or Phomopsis sp. colonies were then placed on minimal medium with nitrate. Nit isolates were identified by their lack of ability to grow normally. Only one nit isolate was recovered from 175 fruit wound-inoculated with Phomopsis sp. In contrast, 21 nit isolates were recovered from 175 fruit wound-inoculated with C. acutatum. Examination of the temperature required for spore germination showed that 50% germination occurred in 24 h at 12.6 degrees C for C. acutatum, and at 11.6 degrees C for Phomopsis sp. Both these temperatures were exceeded for four of the 7 months. In three of those four months, more nit isolates of C. acutatum were recovered.

Bull's eye rot (BER), caused by Neofabraea alba, is an important postharvest apple disease worldwide. Reduction of BER by application of hot water treatments (HWT) was demonstrated in laboratory studies on inoculated 'Scilate' apples. Application of a sanitiser (Tsunami) and UV-C did not provide significant reduction of bull's eye rot, and did not improve HWT when used in combination. Warming fruit after 3 weeks coolstorage followed by replacing fruit in the coolstore reduced bull's eye rot, but not as effectively as HWT. The feasibility of using HWT in a semi-commercial packing line was investigated. One bin of naturally infected ' Scired' fruit was harvested from an orchard with known high incidence of BER, then placed into a coolstore at 0.5 +/- 0.5 degrees C for 1 week until treated. All fruit were passed through a high pressure water washer then air-dried. Half the bin's fruit were packed into cardboard trays in apple boxes with a plastic polyliner. The other half were treated for 2 min with hot water at 51 degrees C in a semi-commercial hot water bath, then air-dried before packing as before. After all treatments were conducted, fruit were placed in a coolstore at 0.5 +/- 0.5 degrees C and assessed after 6, 12, 16 and 20 weeks. After 20 weeks of coolstorage, the hot water-treated fruit showed less lesion growth (identified by symptoms as BER) than untreated control fruit. HWT effectively reduced both lesion size and number of natural infections by Neofabraea alba by 93 and 84%, respectively, and shows promise for use in commercial packhouses.

Abstract The avocado (Persea americana Mill.) is from an ancient plant lineage, the Lauraceae. Although evidence for human consumption dates back 15,000 years, commercialisation has occurred only over the last 150 years. The most commonly traded variety was first the green-skin 'Fuerte' (green as it ripens), and more recently 'Hass', on which skin darkens when ripe. Production has been increasing worldwide, and currently about 64 countries produce avocados. The range of climates is from arid to very high rainfall and from tropical to temperate. The minimum daily temperatures are above 5°C in all avocado-growing regions because of frost sensitivity. Apart from avocado sunblotch viroid (ASBVd), most avocado fruit diseases are caused by fungi. Some fungi cause visible symptoms resulting in unmarketable fruit, and other infections in the orchard are symptomless. These symptomless infections express as rots after harvest during cold storage, transport and ripening. Most post-harvest pathogens infect through both the body of the fruit and the stem-end wound, while a few infect only through the stem-end wound. The geographic distribution of these fungi varies possibly because of differences in environmental requirements and effective quarantine measures during trade. Fungal rots can be reduced by the application of fungicides in the orchard, removing inoculum residing in dead branches and mummified fruit in the canopy, ensuring high-calcium levels in the fruit flesh are maintained, careful post-harvest handling and selling fruit as soon after harvest as possible. Some post-harvest fungicides can be effective.

Molecular detection of phytopathogens is increasingly being applied to identify regulated organisms at the border in many parts of the world. However, even with molecular tests, complete phenotyping and identification of a strain is often time consuming and sometimes inconclusive. In this study, a leaf-based pathogenicity test was used to separate pseudomonads into two groups, Group A containing pathogens, and Group B containing saprotrophs. Comparative genomics of 56 pseudomonad genomes from different plant hosts (including 29 strains from kiwifruit) agreed with kiwifruit pathogenicity test results, placing pathogens into Group A and saprotrophs into Group B. Sixteen loci were found unique to Group A. A PCR assay was developed for amplification of one of these loci, the trehalose phosphatase gene. The generation of this 655 bp amplicon was associated with production of water-soaked lesions on inoculated kiwifruit leaves by pseudomonads in Group A. This test was validated for further strains from all seven pathogenicPseudomonasphylogroups, non-pathogenic pseudomonads, and other bacterial genera. The sensitivity of the PCR was comparable to the limit of recovery of pseudomonads by culturing. This simple PCR assay could be used as part of a testing pipeline at the border and for general surveillance for screening plants with and without symptoms, offering the potential to detect uncharacterized pseudomonads that may pose a biosecurity risk. The method was shown to be able to rapidly identify pathogens cultured from plant material with symptoms, or, more importantly, to detect pathogens directly from plant tissue.

Phlyctema vagabunda syn. Neofabraea alba is a fungal pathogen that causes bull’s eye rot (BER) of apples. Polymerase chain reaction (PCR) primers complementary to the inter-transcribed spacer region of ribosomal DNA (ITS) and the β-tubulin gene region, and a TaqMan™ probe assay were developed to detect this pathogen. These assays were compared in quantitative PCR (qPCR) reactions for amplification of DNA extracted from several fungal species and from apple tissue. Although the ITS and the β-tubulin primers amplified all N. alba isolates, both primers also amplified a few other fungal species. The TaqMan™ probe used with published primers for N. alba only amplified N. alba isolates. The TaqMan™ assay resulted in the lowest crossing threshold (Ct) values for DNA extracted from apple fruit, leaves, and spores collected on cellophane from eight apple orchards. The TaqMan™ results were correlated with percentage BER (%BER) in a 400-apple sample harvested from the same orchards. The TaqMan™ probe assay was the most sensitive and specific qPCR protocol tested, and Ct values showed the best correlation with %BER.

Fungal pathogens are the main cause of postharvest losses of apples, pears, and other pome fruits. The main fungal diseases of pome fruit are considered to be blue mold, caused by Penicillium spp.; grey mold, caused by Botrytis cinerea; bitter rot and other Colletotrichum spp. diseases; bull's eye rot, caused by Gloeosporium spp.; brown rot, caused by Monilinia spp.; and storage apple scab, caused by Venturia inaequalis. Besides these main diseases, some minor diseases are also described, including black rots, soft rots, core rots, and white haze. For each disease, economic importance, main symptoms, available tools for pathogen identification, pathogen biology, and disease epidemiology are illustrated. The tools and strategies available in the orchard and after harvest for the management of postharvest diseases are considered. The last section is dedicated to the most important physiological disorders of pome fruits: scald and bitter pit. For the physiological disorders, information about economic importance, symptoms, etiology, and control are provided.

Bull’s eye rot (BER) of apples is caused by a postharvest fungal pathogen (Phlyctema vagabunda syn. Neofabraea alba). Previous laboratory experiments found hot water treatments (HWT) resulted in a significant reduction of BER incidence for artificially inoculated fruit so the feasibility of HWT to control naturally infected fruit in a semi-commercial trial was tested. One bin (1934 fruit) of naturally infected ‘Scired’ apples was harvested from a Hawke’s Bay orchard with a known high incidence of BER, then placed in a coolstore for 1 week until treated. All fruit were passed through a high-pressure water blaster then air dried. Approximately half the contents of the bin (1034 fruit) were packed into Friday trays in apple boxes with a plastic polyliner. The other half (900 fruit) were treated for 2 min with hot water at 51°C in a semi-commercial hot water bath before packing. All fruit were then coolstored for 20 weeks before assessment for BER. This HWT resulted in a 6-fold reduction of BER incidence so was an effective treatment for BER in a semi-commercial test.

Neofabraea actinidiae (syn. Cryptosporiopsis actinidiae) is a member of a suite of fungi associated with ‘ripe rots’ of kiwifruit. Although it has been recorded regularly from kiwifruit in New Zealand over the past 30-40 years, initially as ‘Cryptosporiopsis sp.’, there is a general lack of knowledge of this fungus. This paper provides a review of the current records and available literature on the taxonomy and biology of the organism, and assesses the knowledge gaps in the disease cycle and epidemiology of N. actinidiae in kiwifruit orchards. The conidia of the fungus are likely to be water borne, infect fruit during or near to flowering and remain latent until harvest and subsequent ripening. The source of inoculum remains unknown. This review may stimulate new research into this pathogen and give insights into potential control strategies.

A variety of pseudomonads are associated with diseases of Actinidia and Prunus plants. A recently emerged virulent haplotype of Pseudomonas syringae pv. actinidiae (biovar 3) causes severe stem cankers on kiwifruit associated with dieback of canes. Pseudomonas syringae also causes one of the most important bacterial diseases affecting cherry orchards worldwide. Bacterial canker of cherry limits production in orchards in New Zealand. Less virulent and non-pathogenic pseudomonads also exist on both hosts, providing an opportunity to investigate the diversity of pseudomonads on these hosts, to find identifiers for new emerging highly pathogenic strains that could be used at the border to prevent incursions. In this study, genetic typing was used to explore the diversity of Pseudomonas on kiwifruit and a variety of Prunus plants. Multilocus sequence analysis (MLSA) of four house-keeping genes separated pseudomonads from kiwifruit and stone fruit plants into two major groups: one corresponding to P. syringae sensu lato and one corresponding to other Pseudomonas species. Within P. syringae sensu lato, strains were assigned to six of the nine previously described genomospecies or five of the seven previously described phylogroups. In the other major group, strains from both hosts clustered with a variety of well characterised non-pathogenic pseudomonads. The classification of strains into the two major groups is of practical diagnostic value since the most common pathogens of fruit trees belong to the P. syringae species complex. Furthermore, our analysis suggests that molecular diagnostics might be possible for the classification of strains into these two groups as a first tool to screen for exotic pathogenic pseudomonads on germplasm imports of both commodities.

A virulent strain of Pseudomonas syringae pv. actinidiae (Psa) is a major pathogen for New Zealand’s $3B kiwifruit (Actinidia spp.) industry, and was first identified from a Te Puke orchard on 5 November 2010. Psa was first found on the Kerikeri research orchard (KRO) of Plant & Food Research on 19 September 2014. The samples for this study were collected from the same orchard on 7 December 2012 and 25 November 2014, i.e. before and after the Psa incursion. Polymerase chain reaction (PCR) was conducted on total genomic DNA from four leaf discs of 15 individual vines sampled from two kiwifruit cv. ‘Hort16A’ orchard blocks at KRO, using modified PCR primers complementary to bacterial 16S ribosomal DNA and the fungal inter-transcribed spacer (ITS) region. The microbiota present before and after the Psa incursion were investigated by Illumina MiSeq™ next-generation sequencing to produce 2 × 300 bp pair end reads, followed by metabarcoding analysis using QIIME2 software. Populations of fungi from the Basidiomycete orders Filobasidiales, Sporidiobolales, Tremellales and Leucosporidiales, and genera of bacteria with known biological control activity, such as Erwinia, Pantoea, Methylobacterium, Sphingomonas and Paenibacillus, increased in the presence of Psa.

The avocado industry was established in New Zealand from several importations dating back to 1907. Several serious pathogens found elsewhere in the world were not imported. A literature review and internet search were conducted to determine what serious avocado pathogens are not present in New Zealand and the potential impact they could have if they established. Relevant information was summarised for six pathogens determined to be the most serious of avocado and not known to be present in New Zealand: avocado sunblotch viroid (ASBVd); Pseudocercospora purpurea (cercospora spot); Raffaelea lauricola (laurel wilt); Fusarium sp. (fusarium dieback); Phellinus noxius (brown root rot); and Sphaceloma perseae (avocado scab). Laurel wilt, brown root rot, cercospora spot and fusarium dieback could become established in New Zealand if the climate here becomes warmer but establishment of ASBVd and avocado scab (which are not restricted to hot climates) is more likely.

Neofabraea actinidiae can occasionally cause post-harvest rot in kiwifruit. Quantitative polymerase chain reaction (qPCR) analysis represents a feasible and accurate option for identifying and quantifying this rot but is limited because qPCR results do not differentiate live and dead conidia. Propidium monoazide (PMA) is a photoreactive dye that penetrates into the damaged cell-wall membranes of dead conidia binding to the DNA and thus suppressing its amplification by qPCR. A commercial kit containing PMA was trialled for differentiating between live and dead N. actinidiae conidia. The most suitable conditions were 1 μM PMA with 10 min light emitting diode (LED) exposure, and could clearly distinguish high concentrations of live from similar concentrations of dead conidia when tested separately and as a mixture. Low concentrations of live N. actinidiae conidia could be distinguished from dead ones when tested separately, but not as a mixture. Additional work is needed to optimise the effectiveness of the PMA binding and apply this concept in the orchard.

A method was developed to inoculate and establish infection of detached apple fruit with the bull’s eye rot pathogen, Phlyctema vagabunda (syn: Neofabraea alba), without wounding. Mycelial cultures of P. vagabunda did not produce conidia on commonly used potato dextrose agar, or several other tested media. Growth on corn meal agar resulted in the highest conidial yield, and maximum production was achieved after 5 days. These conidia were placed on water agar, and apple fruit were placed on these spores for at least 5 days for establishment of infections. Reliable infection of 66—100% of apples required 14 days of contact. The ability to rapidly produce copious quantities of conidia and inoculate without wounding facilitates a number of other postharvest control and epidemiology studies.

Current methods for producing conidia of Neofabraea alba the fungal cause of Bulls eye rot of apple are laborious and timeconsuming A minimum of 6 weeks is required Mycelial cultures of N alba did not produce conidia on commonly used potato dextrose agar and therefore several other media were tested Growth on corn meal agar resulted in a high conidial yield (ca 106 conidia/ml) after 57 days growth Published research reports a method to inoculate apples with this pathogen without wounding but it requires specialised equipment A simple method that does not require specialised equipment was developed to inoculate apple fruit with the Bulls eye rot pathogen without wounding Conidia were placed on water agar and apple fruit were placed on these spores for at least 5 days for establishment of infections Reliable infection of 7080 of apples required a 14day wetness period after the fruit were placed on the conidial solution This woundfree N alba inoculation method provides the basis for further research to examine the effect of various treatments designed to reduce the impact of this pathogen in apple orchards

Fruit tree canker, caused by the fungus Neonectria ditissima, is considered to be a serious economic problem in apple orchards, especially in north-western Europe. This fungus produces conidia and ascospores. They are dispersed and cause infection and eventually life-threatening cankers during prolonged periods of rainy weather. Moreover, spores produced on the infected wood can act as an infection source in the orchards and spread easily during the year. Preventing the spread of the fungus is important, and therefore a fast, sensitive and reliable method to detect N. ditissima in infected apple trees would be desirable. A quantitative PCR (qPCR) method was developed for the detection and quantification of N. ditissima in infected apple cultivars. Primers, specific for N. ditissima, were designed and used for qPCR analysis on genomic DNA (gDNA) extracted from infected trees. N. ditissima gDNA was detected at variable amounts in the samples from the infection sites of different cultivars. This quantification method proved to be very accurate, since the relative quantities of gDNA were correlated to the lesion size measured after artificial inoculation of these apple cultivars. The results demonstrate that the qPCR assay is a sensitive method for detection of N. ditissima, and that it could be very useful for screening levels of resistance in germplasm collections.