Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.

Botryosphaeriaceae species are acknowledged as important wound pathogens of grapevines worldwide but reports as to the relative pathogenicity of species have varied. This study showed that the multiple vineyard isolates of Neofusicoccum luteum and N. parvum tested all caused disease of different grapevine stem tissues, but with great variation in symptoms and conidial release between isolates of each species. Overall, N. luteum caused longer lesions and produced more conidia than N. parvum on detached soft and semi-hard green shoots and on soft shoots of potted grapevines as well as on hard shoots of mature vines. With Sauvignon blanc, longer lesions and more conidia were produced on semi-hard green shoots and trunks of potted vines and on semi-hard green shoots of mature vines, than with Pinot noir. All isolates of N. luteum and N. parvum released conidia from stem lesions at all temperatures (10–25 °C) and RH (80–100%) tested, with most isolates releasing greatest numbers in high RH (~100%) and temperature (25 °C). This study showed that the two most commonly found species in New Zealand, N. luteum and N. parvum, were able to infect grapevine tissues and produce conidial inoculum under the temperatures and humidities commonly found in New Zealand vineyards.

Pycnidial and conidial production by isolates of the four main Neofusicoccum species associated with blueberry in New Zealand was investigated. Pycnidia developed after 8 days on mycelial-inoculated detached green shoots. Conidial ooze was observed after further incubation for 12 h under high relative humidity at 25˚C. Numbers of oozing pycnidia and conidial numbers were generally low, but were significantly affected by isolate and species. Neofusicoccum ribis and N. parvum produced slightly more pycnidia and conidia compared with N. luteum and N. australe. Inoculation of non-wounded and wounded attached green shoots with either N. ribis or N. australe conidia showed that, 14 days after inoculation, lesions developed in wounded shoots only, with N. ribis (58.8 mm) producing longer lesions than N. australe (29.8 mm). Neofusicoccum ribis and N. australe were re-isolated beyond the lesion, with pathogen progression being significantly greater for wounded (47.1 mm) compared with non-wounded shoots (30.4 mm).

Genotypic and virulence diversity of Neofusicoccum luteum and N. australe isolates recovered from grapevines displaying symptoms of dieback and decline in New Zealand were investigated. The universally primed PCR (UP‐PCR) method was used to investigate the genetic diversity of 40 isolates of N. luteum and 33 isolates of N. australe. Five UP‐PCR primers produced a total of 51 loci from N. luteum and 57 from N. australe with a greater number of polymorphic loci produced in N. australe (86%) compared with N. luteum (69%). Analysis of UP‐PCR data showed both species found in New Zealand vineyards were genetically diverse at both the inter‐ and intra‐vineyard levels with only a single pair of clonal isolates in N. luteum. Cluster analysis of UP‐PCR data produced four genetic groups in N. luteum and 10 in N. australe (P < 0.05). For both species, there was no relationship between the genetic groups and the origin of isolates. The mean genetic diversity (H) of N. luteum was less than for N. australe, being 0.1791 and 0.2417, respectively. Pathogenicity assays of both species using isolates from either the same or different genetic groups inoculated onto either green shoots or grapevine trunks, showed virulence diversity within the population; however, no correlation was identified between genetic groups and virulence.

Botryosphaeriaceae species are important pathogens of grapevines so the effect of environmental conditions on sporulation from naturally infected Sauvignon blanc grapevines in two Marlborough vineyards was studied. Shoot lesions characteristic of Botryosphaeriaceae infection were marked on 24 grapevines in each vineyard for observation between September 2014 and April 2015. Pycnidia on the lesions oozed conidia during or soon after rainfall on five occasions at maximum air temperatures of 8.4—19.9˚C and relative humidities of 77—94.7%. Microscopic observation of the ooze showed conidia characteristic of either Neofusicoccum or Diplodia species in 67% and 50% of samples from Vineyard A and B, respectively. Sixty- nine colonies characteristic of Botryosphaeriaeae species were recovered after plating of conidial ooze and identified by DNA analysis as Diplodia mutila (39.1%), Neofusicoccum australe (30.4%), N. parvum (14.5%), N. luteum (5.8%), D. seriata (7.2%) and N. ribis (3.0%). All species have been reported previously as grapevine pathogens in New Zealand.

Species of Botryosphaeriaceae are important wound pathogens of grapevines as causal agents of botryosphaeria dieback, but the behaviour of their conidia pre‐infection is unknown and may be important for disease development. Adhesion properties of conidia were investigated for Botryosphaeria dothidea, Neofusicoccum luteum and N. parvum on substrata with different affinities for water. Greatest adhesion on any surface was reached after 5 min for isolates N. luteum MM558, B. dothidea 007 and N. parvum G652 (53·1, 54·0 and 50·6%, respectively) and for N. luteum isolate CC445 after 20 min (61·4%). As conidia adhered well to all artificial substrata, it appeared as if the attachment process was nonspecific. Overall, surface wettability did not play a major role in the adhesion of conidia. Spore surface proteins appeared to play a role in the adhesion process because treatment of conidia of N. luteum MM558 with a protease completely prevented adhesion. Histochemical labelling of conidia and germlings with Coomassie brilliant blue (specific for proteins) was positive for all isolates, with a blue ‘halo’ often seen surrounding conidia or near the germ tube emergence point after incubation times conducive to germination. Alcian blue also stained material surrounding conidia after longer incubation times, which indicated that mucopolysaccharide and protein production may be involved in a second phase of adhesion.

Downy mildew of boysenberry caused by Peronospora sparsa is a major disease problem for New Zealand growers Investigation of the biology and epidemiology of this biotrophic pathogen requires the production of sporangiospore inocula Four leaf sterilisation treatments (70 ethanol 10 bleach sterile water control) were assessed for their ability to reduce surface contaminants After treatment the leaf surfaces (six replicates) were pressed onto potato dextrose and nutrient agar plates and these were incubated at 20C for 1 week All treatments significantly (P

Botryosphaeria dieback of blueberry is caused by several botryosphariaceous species including Neofusicoccum ribis This research investigated whether stem damage caused by three herbicides could allow for penetration of blueberry stems by this pathogen Results showed that all the herbicides damaged hard green shoots of which 100 were penetrated by the inoculated N ribis Mean lesion lengths were similar for all herbicides and the inoculated wounded control (P0240) Isolations from bark and wood showed similar infected proportions for bark (P014) but different proportions for wood (P0035) These mean proportions were 764 for bark and 825 for wood when treated with glufosinateammonium 746 and 750 with paraquatdiquat 669 and 614 with carfentrazoneethyl and 581 and 604 for the inoculated wounded control This study has indicated that herbicide damage can create entry ports for stem pathogens and highlights the importance of careful herbicide application to manage understorey weeds in blueberry farms

Botryosphaeriaceae species cause dieback and canker in many woody hosts including grapevines with infection occurring when conidia are released during rainfall and splash borne to pruning and trimming wounds This study monitored dispersal of naturally released conidia of Botryosphaeriaceae species in three Marlborough vineyards with a Burkard spore trap and rain water traps Microscopic examination of the Burkard tape and trapped rain water confirmed the presence of Neofusicoccum and Diplodia spp Species were identified on tape and in rainwater with single stranded conformational polymorphism as N luteum N parvum/Nribis N australe D mutila and D seriata To determine conidium dispersal distances sporulating shoot lesions of N parvum isolate B2141 for which an isolate specific marker was developed were placed in one Marlborough vineyard before forecast rainfall periods The rainwater traps were set up around the sporulating lesions at 05 to 20 m in the direction of the prevailing wind and 05 to 5 m in three other directions After 2 days rain Neofusicoccum sp conidia were identified in the collected rainwater by microscope and with the isolate specific PCRRFLP (restriction fragment length polymorphism) for N parvum B2141 This isolate dispersed up to 10 m in the wind direction and up to 1 m in the other three directions

Past studies have shown that the major source of infection of young grapevine plants by Botryosphaeriaceae fungi were through the use of infected rootstock and scion cuttings. To investigate the potential infection pathways of Botryosphaeriaceae species within a rootstock mother vine, three genotyping studies using universally-primed polymerase chain reaction (UP-PCR) were conducted with two Neofusicoccum species, namely N. luteum and N. parvum. The investigations identified genotypes of the fungal isolates in trunk and shoot infections of the same mother vine. Results showed that the trunk and shoot isolates from the same vine were of the same or different genotypes, suggesting multiple infections from different inoculum sources. This study further showed that the Neofusicoccum isolates recovered from the surfaces of the cuttings were of the same or different genotypes from those isolated from adjacent internal tissues, again suggesting multiple sources of external inoculum. Investigations into the spatial distribution of Botryosphaeriaceae fungi within an entire dormant cane also showed that multiple species and genotypes were distributed along the cane but most isolates were sited within the bark, being less frequently in the wood, which suggested that they were latent on surface tissues. Since some adjacent wood and bark infections were caused by the same genotypes, this indicated that wood infection may have originated from the bark. These fungi appear to cause latent infections in the bark of dormant cuttings which are used in plant propagation, thus providing an additional infection pathway for a disease that is known to show obvious symptoms only in older vineyards.

This study investigated the susceptibility of the most common rootstock and scion varieties used in New Zealand grapevine nurseries and vineyards to Botryosphaeriaceae species. A total of six rootstocks and six scion varieties were inoculated with mycelium of three isolates each of the most common Botryosphaeriaceae species (Neofusicoccum luteum, N. parvum and N. australe) recovered from New Zealand grapevine nurseries. Overall results showed that all isolates produced necrotic lesions on all varieties but susceptibility varied significantly (P < 0.001) with 5C and SO4 being the most susceptible of the rootstock varieties, and Merlot and Pinot noir being the most susceptible of the scions. Pathogenicity also varied significantly (P < 0.001) between species with N. parvum being the most virulent among three species tested. This study has further shown that different isolates of the three species have different levels of virulence providing evidence that these pathogens are genetically diverse. These results have significant implications for the New Zealand grapevine industry as the use of resistant varieties is not a possible option at present since none of the common varieties are resistant to these pathogens.

Species within the Ilyonectria macrodidyma complex are known plant pathogens and several are implicated as the causal agents of black foot disease of grapevines The seven species within the complex can be identified by DNA sequencing of the histone H3 gene In this study a PCRRFLP method to identify the species was developed In silico digestion of the 500 bp histone H3 amplimer using MnlI showed that it could identify Ilyonectria sp 1 Ilyonectria sp 2 I alcacerensis and I macrodidyma Subsequent in silico digestion with Hinf1 identified I estremocensis I novozelandica and I torresensis The PCRRFLP was validated using a collection of 40 I macrodidyma complex isolates that had been recovered from symptomatic grapevines Ilyonectria macrodidyma I novozelandica and I torresensis were detected in that collection Intraspecific polymorphism was only detected in I torresensis This method provides a rapid procedure for identifying individual species of the I macrodidyma species complex

A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species-specific PCR and DNA sequencing from a collection of 174 Ilyonectria-like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP-PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour-joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra- and inter-vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non-self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1-year-old potted vines showed variability in virulence; however, no correlations were detected.

Several botryosphaeriaceous species have been reported to cause stem canker twig blight and dieback of blueberries with different Botryosphaeria species reported in different parts of the world This research evaluated nine fungicides for their ability to reduce mycelial growth conidial germination and germ tube elongation of four common and pathogenic botryosphaeriaceous species recovered from New Zealand blueberries Furthermore two biocontrol agents were tested against the same species using dual plate assays Fludioxonil carbendazim flusilazole and tebuconazole were the most effective for inhibition of mycelial growth of three isolates each of Neofusicoccum australe N luteum N parvum and N ribis EC50 values for these fungicides were less than 01 mg ai/litre Carbendazim and iprodione (both EC50 004 mg/litre) were the most effective for reduction of conidial germination of all four species In addition these two fungicides were effective for inhibition of germ tube elongation with mean EC50 values of 004 and 01 mg/litre respectively The biocontrol agents Trichoderma atroviride and Bacillus subtilis reduced mycelial growth of all species tested with a range of macroscopic interactions This study has indicated the most promising fungicides and biocontrol agents for further investigations to protect pruning wounds in blueberries

This study investigated some factors that affect Neofusicoccum luteum infection and disease progression in grapevines, on which it causes cankers and die-back. Inoculation of green shoots with each of the conidial suspensions (20 μL of 102–106 conidia/mL) caused 100 % infection, but significant differences in lesion lengths among concentrations (P < 0.001). Lesion lengths increased with conidial concentrations to a maximum of 41.7 mm, in tissue inoculated with 104/conidial. Lesions then decreased for 105 and 106 conidial/mL (28.6 and 27.8 mm, respectively). The different wetness durations (0–24 h) tested after inoculating trunks of 1-year old vines with conidia did not affect pathogen incidence or lesion lengths, however continued incubation at 95 % RH caused greater pathogen progression (maximum isolation distances from point of inoculation; P < 0.001; 100.6 mm) than at ambient 78 % RH (77.0 mm) within 3 months after inoculation. Inoculation of trunks, canes and green shoots caused different rates of pathogen colonisation (P < 0.001); by 5 months after N. luteum inoculation mean maximum distances were 39.1, 31.6 and 23.2 mm, respectively. Wound age at the time of inoculation significantly affected (P < 0.001) infection incidence by N. luteum, which decreased as wounds aged. Conidial inoculum caused 100-0 % incidence over 0–14 days and mycelial inoculum caused 100-40 % infection over 0–30 days. Soil moistures affected lesion development, with 15 and 100 % soil moisture causing greatest dieback and bud death during winter. This study has shown how some features of the natural infection environment can affect infection by Botryosphaeria spp. inoculum and symptom development, which could underpin improvements in vineyard management.

Phoma black leg or stem canker, caused by Leptosphaeria maculans or L. biglobosa, is an important disease of brassicas, causing significant crop losses in areas such as Europe, Australia, and North America (1). Samples collected in 2011 from canola and forage brassica (swede, kale, and turnip) crops in the main New Zealand growing regions (Southland, Central Otago, Canterbury, Hawkes Bay, and Manawatu) to identify the causal agent(s) of the characteristic stem cankers, found many isolates of L. maculans, which has been reported previously in New Zealand (2), and three isolates identified by colony characteristics as L. biglobosa. Of the latter, two isolates were from canola (Brassica napus) stem cankers from Darfield and Lincoln, Canterbury, and one was from a kale (B. oleracea) stem canker from Lincoln. An isolate (ICMP10665) of similar morphology, from the International Collection of Microorganisms from Plants (ICMP), obtained from a basal rot lesion on a cauliflower (B. oleracea var. botrytis) plant in Levin, New Zealand in 1979, was also evaluated. The initial, incorrect identification of the latter isolate as L. maculans predates the reclassification of L. maculans group B isolates as a new species, L. biglobosa (1). These four isolates produced fluffy white mycelium and a yellow pigment on potato dextrose agar (PDA) after 5 days' growth, and abundant black-brown, globose pycnidia containing cylindrical hyaline conidia after 7 days. In contrast, L. maculans isolates had slower growth and no pigment production (4). Amplification of genomic DNA using species-specific primers LmacR, LmacF, and LbigF (1) generated a PCR product of 444 bp that is typical of L. biglobosa isolates. Sequencing of the PCR product from each of the four isolates showed they were 100% identical to a sequence of L. biglobosa ‘brassicae’ in GenBank (JF740198). To confirm the species identity of the isolates, the rDNA, actin, and β-tubulin gene regions were amplified (1,3). Sequences for the rDNA (568 bp), actin (941 bp), and β-tubulin (410 bp) gene regions were 99% identical to sequences of the same regions of isolates in GenBank for L. biglobosa ‘brassicae’ (AY48997, AY748949.1, and AY748997.1, respectively). The four L. biglobosa isolates were tested for pathogenicity on a canola cultivar commonly grown in New Zealand (Flash). Cotyledons of 10-day-old seedlings (n = 12 seedlings/isolate or control treatment) grown in a potting mix in pots were pricked with a sewing needle, and each wound inoculated with 10 μl of the appropriate conidial suspension (106 conidia/ml) or 10 μl sterilized distilled water for the control treatment. Leaf lesions that developed on the inoculated cotyledons were characteristic of those caused by L. biglobosa, i.e., small and dark with a distinct margin. No pycnidia were produced on the lesions. No lesions developed on the cotyledons of the non-inoculated control plants. The causal agents were confirmed as L. biglobosa by the colony morphology of isolates that grew from surface-sterilized, inoculated leaf lesions plated on PDA amended with 100 μg/ml ampicillin. The fungus was not isolated from control leaf tissue. To our knowledge, this is the first report of L. biglobosa as a pathogen of canola and kale in New Zealand. This finding shows that both causal agents of black leg are present in New Zealand's brassica cropping areas. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) H. C. Smith and B. C. Sutton. Trans. Brit. Mycol. Soc. 47:159, 1964. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) R. H. Williams and B. D. L. Fitt. Plant Pathol. 48:161, 1999.

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP‐PCR) analysis of isolates obtained from grapevines and non‐grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR‐RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate‐specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate‐specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate‐specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event.

The introduction of Gaeumannomyces graminis var. tritici (Ggt) inoculum into soils to screen for take-all suppression has been widely used in field and laboratory studies. However, the amounts of Ggt inoculum reported have varied greatly. The effects of adding Ggt in sand/maizemeal to three soils of different cropping history at the rates of 0, 0.2, 0.5, 1 and 4 % (w/w) were investigated in a pot assay using wheat plants. The three soils had previously been cropped with ryegrass for 5 years, wheat for 8 years, and wheat for 2 years. The soils represented a putative non-suppressive, non-wheat soil; a suppressive wheat soil; and a non-suppressive wheat soil, each containing natural background concentrations of Ggt DNA of 0, 200 and 1126 pg g−1 soil, respectively. Root assessments of wheat plants after 4 weeks growth showed that 4 % of Ggt reduced root growth slightly, decreased water uptake of the wheat plants and effectively differentiated the suppressive activity of the soils (P < 0.01, take-all incidences of 83, 69 and 81 %, respectively), and was therefore suitable for investigating take-all suppression in soils.

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.