Pathogenicity and progression of Botryosphaeriaceae associated with dieback in walnut orchards in Australia

Dieback of the fruiting spurs of wal-nut ( Juglans regia ) results in yield losses in walnut orchards in Australia. Botryosphaeriaceae spp. have been implicated as the causative agents, with Dip‑ lodia seriata and Neofusicoccum parvum reported as the most prevalent species. Pathogenicity assays demonstrated inter-and intra-species variation with N. parvum more virulent than D. seriata . Both species were re-isolated beyond the dieback lesion. At 12 months post-inoculation on one-year-old trunks, N. parvum and D. seriata were re-isolated at 31.5 and 9 mm respectively beyond lesion edges. The lesion length as well as pathogen progression beyond the lesion increased over time. The pathogen progressed faster acropetally than basipetally. Following wound healing at the site of inoculation, and in the absence of any external lesion, both pathogens continued to move through the xylem, causing internal discolouration of the vascular tissues. Both mycelia and conidia were pathogenic, with mycelia showing higher infection rates. There was a strong linear relationship between the dieback length caused by the two types of inocula. As little as two conidia were able to infect wounded tissues. Although wounds were susceptible to infection for more than 4 months post-wounding for N. parvum and 1 month for D. seriata , the highest disease incidence occurred in the first week following wounding. The implications for the walnut industry for appropriate control strategies are discussed.


Introduction
The tree nut industry provides a significant contribution to Australia's horticultural exports, accounting for more than a third of its export value (Australian Nut Industry Council, 2022).Within this expanding industry, walnuts are the third largest sector, next to almond and macadamia.An emerging challenge to the productivity of the walnut industry is yield loss due to dieback of fruiting spurs.Symptoms of dieback, cankers, blight and fruit rot have been reported in walnut orchards in a number of countries, including Chile (Luna et al., 2022), China (Yu et al., 2015;Zhang et al., 2017;Li et al., 2023), Egypt (Haggag et al., 2007), Iran (Sohrabi et al., 2020), Italy (Gusella et al., 2021), Spain (López- Moral et al., 2020b), Turkey (Yildiz et al., 2022) and the USA (Michailides et al., 2012).Species of Botryosphaeriaceae have been implicated in these symptoms.
In Australia, Burgess et al. (2019) reported the occurrence of 62 Botryosphaeriaceae species, after verifying the publicly available DNA sequences.At least 20 of these have been identified as causal agents of disease in horticultural crops in Australia.Lasiodiplodia iraniensis, L. pseudotheobromae, L. theobromae, Neofusicoccum australe, N. luteum, and N. parvum in macadamia (Jeff-Ego & Akinsanmi, 2019), and Botryosphaeria dothidea in pistachio (Wunderlich et al., 2012) have been linked to economic loss in nut crops.In walnuts, the incidence of Dothiorella omnivora, Do. vidmadera, Diplodia seriata, N. australe, and N. parvum have been reported by plant diagnostic laboratories where samples have been sent for diagnostic analysis (Aldaoud et al., 2017;Lang, 2017;Lang & Simpson, 2018).The pathogenicity of these species was not investigated further until 2019 when a systematic survey was initiated.This first survey of walnut dieback in Australia was conducted across the major walnut growing regions during the 2019-2020 growing season.Diplodia seriata, Do. omnivora, N. macroclavatum, N. parvum and Spencermartinsia viticola were isolated, taking the number of species of Botryosphaeriaceae reported in walnuts in the country to seven (Antony et al., 2023).The survey also identified D. seriata and N. parvum as the most prevalent species in the walnut orchards surveyed in Australia.
Pathogenicity tests conducted by Antony et al. (2023) on detached one-year-old stems of walnuts showed that, except for Do.omnivora, all the other four species caused lesions that were significantly longer than the control (P<.05), with N. parvum causing lesions three times greater than D. seriata.One of the critical factors linked to infection by species of Botryosphaeriaceae in woody crops is the effect of wounds and age of wounds.Wounds are regarded as primary infection sites for Botryosphaeriaceae species in grapevines (van Niekerk et al., 2004), walnuts (Adaskaveg et al., 2017) and almonds (Olmo et al., 2017).However, in walnut orchards, wounding the trees is unavoidable.During the growing season several orchard management practices such as training young trees, pruning older trees for canopy management and mechanical harvesting cause wounds to the trees.An understanding of how long these wounds are susceptible to infection and any other factors that increase the risk of infection is essential to protect the health of the trees.Studies on other crops have shown that disease progression is also dependent on the amount of inoculum (Amponsah et al., 2014;Sosnowski et al., 2017).Against this background, in this study some of the factors that predispose walnut trees to dieback were elucidated, so that future disease incidence and severity can be minimised.For the two prevalent species namely D. seriata and N. parvum, the following questions were investigated: Is there inter-and intra-species variation in the pathogenicity of the prevalent species?How far beyond the visible internal dieback symptoms do these species colonise the wood?What is the effect of inoculum type and inoculum concentration on the severity of infection?What is the effect of wounds on disease incidence and for what time period are wounds susceptible to infection?

Selected isolates and inoculum preparation
Botryosphaeriaceae isolates recovered from walnut orchards during a previous study were used in all mycelial and conidial inoculations.The most virulent isolates of D. seriata (DS04, DS15, and DS27) and N. parvum (NP03, NP05, and NP18), were selected based on their previously determined virulence and lesion length that they caused on detached stems (Antony et al., 2023).For mycelial inoculation, mycelial plugs stored at 4 °C were sub-cultured onto potato dextrose agar (PDA; Oxoid Ltd., UK) and incubated at 25 °C in darkness for 3-5 days.For conidial inoculation, the selected isolates were grown on prune extract agar (Antony et al., 2023).To prepare the conidial suspension, the agar plate containing pycnidia were flooded with 10 mL of sterile distilled water (SDW) containing 0.01% (v/v) of Tween 80 (G-Biosciences, USA), and the pycnidia dislodged from the agar with a sterile scalpel.The pycnidia and wash water were aseptically placed into a sterile mortar and crushed with a pestle to release the conidia.The liquid suspension was filtered through two layers of Miracloth ® (Merck, Darmstadt, Germany) and standardised to the required concentration using a haemocytometer.To make the conidial suspension of a species, an equal volume of the filtered conidial suspension from each of the three selected isolates was combined in a sterile tube.The conidial suspensions were kept in an insulated box with ice packs during preparation and used within 2 h of preparation in laboratory and glasshouse experiments.Conidial viability for each experiment was confirmed by plating 100 μL of the conidial suspension at 10 4 , 10 3 and 10 2 conidia/mL concentrations, in PDA amended with Triton X-100 (Sigma-Aldrich TM , USA) (1 mL/L), with three plates per species per concentration.All plates were incubated at 25 °C for 5-7 days and colony forming units were counted.

Pathogen inoculation and re-isolation
Mycelial plugs of individual isolates were used for inoculation in the glasshouse experiment that investigated inter-and intra-species variation in pathogenicity and pathogen progression.The other experiments primarily used conidial inoculations, and mycelial inoculations were included in the experimental design when the type of inoculum itself was investigated as part of the experiment.Single isolate suspensions of conidia were used in the experiment on inoculum type.In all other experiments, mixed-isolate suspensions of conidia were used.
All detached stem and shoot experiments were conducted on apparently healthy walnut tissues (cv.Chandler) collected from an orchard where dieback symptoms were not observed.One-year-old stems (~30 cm in length and 15 mm in thickness) were collected in late winter and the green shoots (~30 cm in length and 10 mm in thickness) were collected in late spring.The stems and shoots were surface sterilised by soaking in 2% (v/v) sodium hypochlorite for 2 min, followed by rinsing twice in SDW.For inoculations at the mid-section of the stems and shoots, the inoculation site was further sterilised by spraying with a 70% (v/v) ethanol solution and air dried in a biohazard cabinet for 5 min.A 3-mm diameter wound was made into the one-year-old stem using a sterile drill-bit while a sterile scalpel was used for green shoots.For mycelial inoculations, a mycelium plug obtained from the actively growing margin of a fungal colony growing on PDA was placed in the wound and wrapped with Parafilm ® (Bemis, USA).For conidial inoculations at the mid-section of the stems and shoots, a cup like structure was formed around the site of inoculation with Parafilm ® (Fig. 5 a) to contain the conidial suspension.After inoculation, the inoculated area was wrapped with Parafilm ® to reduce evaporation and the top end of the stem and shoot was also covered with Parafilm ® to avoid desiccation.For inoculations at the tip of the stems and shoots, after surface sterilisation, the tips were pruned, and the conidial suspensions were applied.The inoculated stems and shoots were separately placed into 125 mL plastic tubes filled with SDW.The tubes were arranged in a randomised complete block design (RCBD), and kept in an enclosed transparent plastic chamber that had a thin layer of water at the bottom covered with wet tissue papers, to provide high humidity for 24 h.Stems and shoots were maintained at ambient temperature of 17-25 °C for 4 to 6 weeks in a glasshouse, and tubes were refilled with water, as necessary, during the incubation period.
For the experiments on potted trees, one-year-old walnut plants (cv.Chandler) from a commercial nursery were grown in a glasshouse for 6 to 20 months in 45 L grow bags.The inoculation site was sterilised with 70% ethanol as per the detached stem assay.To inoculate the trunk, a 5-mm diameter and ~3-mm deep wound was made into the trunk using a sterile drill-bit.For the one-year old stems and green shoots, wounds were made at the inoculation site as described previously for the detached stems and shoots.For the conidial inoculation at the pruned tip of the stem and shoot, the treated stem and shoot was covered with a transparent plastic bag for 24 h to prevent evaporation of the conidial suspension.The inoculated plants were maintained in the glasshouse at ambient temperature of 17-25 °C.
Fungal re-isolations were made from the margins of the lesions to confirm Koch's postulates.After surface sterilisation, small pieces of wood were cut aseptically at the lesion margin and transferred to PDA plates that were incubated at 25 °C in darkness.After 3-5 days of incubation, the emergence or absence of fungal growth characteristic of the Botryosphaeriaceae species was recorded.

Pathogenicity in glasshouse plants
Variation in pathogenicity and progression beyond the lesion of N. parvum and D. seriata isolates were investigated in this experiment using glasshouse plants.Walnut plants grown in the glasshouse for 6 months, were allotted seven inoculation treatments (six isolates selected above and one control) with six replicates in a RCBD.After sterilising the site of inoculation in the middle of the trunk and making a wound, mycelial inoculations were performed as described above.The control plants were inoculated with PDA only and the inoculated plants were maintained in the glasshouse at ambient temperature of 17-25 °C.Twelve months after inoculation, the plants were assessed for lesion development.The central trunks of the plants were cut close to the crown and the bark was removed from the entire trunk to assess the extent of the lesions.The trunks were sectioned longitudinally in half through the point of inoculation and internal and external discolouration of the wood (lesions) was measured with a digital calliper.Differences in lesion lengths between isolates and species were compared using analysis of variances (ANOVA) in IBM SPSS Statistics for Windows (Version 27.0.Armonk, NY: IBM Corp).
Fungal re-isolations were made from the lesion margins, to confirm Koch's postulates.To determine the degree to which fungi had colonised the trunk, pathogen re-isolation was also done at 10 mm intervals from the point of inoculation up to 100 mm above and below the lesion edges.The greatest distance of re-isolation above and below the lesion edges was then cumulated to determine total colonisation distance.Differences in colonisation distance between isolates and species were compared as above for the analysis of lesion lengths.To monitor the progression of the pathogen into the side shoots, pathogen re-isolation was also done for the side shoot closest to the point of inoculation that was still alive, at 10 mm intervals up to 40 mm from the base of the shoot.The relationship between lesion length and pathogen progression beyond the lesion was analysed using paired sample t-test, in IBM SPSS Statistics for Windows (Version 27.0).

Infection potential of mycelia and conidia
The effect of inoculum type (mycelial and conidial), of the isolates of N. parvum and D. seriata assessed in the previous experiment was studied using detached one-year-old stems and green shoots.As previously described, asymptomatic one-year-old walnut stems were inoculated in the mid-section of the stems.For the mycelial inoculation, a 3-mm diameter mycelial plug obtained from an actively growing margin of a fungal colony growing on PDA was placed on the wound.The negative control stems were inoculated with a PDA plug only.For conidial inoculation, single-isolate conidial suspensions (10 4 conidia/mL) were prepared for the six isolates as described above, and a 50 μL conidial suspension was applied to each inoculation site.The inoculated stems were placed in plastic tubes filled with SDW, arranged in a RCBD with six replicates, exposed to high humidity for 24 h, and maintained at ambient temperature of 17-25 °C in a glasshouse for 6 weeks.A similar experiment was conducted using apparently disease-free green shoots collected in spring from the same orchard and assessed after 4 weeks.
On completion of the experiments, stems and shoots were removed from the plastic tubes, the bark/dermal tissue was peeled, and the internal and external lesions measured.Fungal re-isolations were made from the margin of the lesions, as in previous experiments, to confirm Koch's postulates.Differences in lesion lengths and the relationship between the lesion lengths caused by the two types of inocula were analysed using ANOVA and paired sample t-test as described above.

Effect of inoculum concentration on disease incidence
The infection potential of different concentrations of conidia, from 2 to 5000 conidia of D. seriata and N. parvum, was studied using detached one-year-old stems, and green shoots.Two mixed-isolate conidial suspensions of 10 5 /mL concentration were prepared, as described above, and used for a dilution series containing 10 4 , 10 3 , and 10 2 conidia/mL.Asymptomatic one-year-old stems of walnut were surface sterilised, pruned at the tip, and pruning wounds were immediately inoculated with conidial suspensions of either D. seriata or N. parvum containing approximately either 5000,2000,500,200,50,20,5 or 2 conidia using sterile micropipettes.Control stems were inoculated with 50 μL of SDW.For each treatment, there were six replicates.As described previously, the inoculated stems in plastic tubes filled with SDW were arranged in a RCBD, exposed to high humidity for 24 h, and then maintained in a glasshouse at ambient temperature of 17-25 °C.
After 6 weeks, the stems were removed from the plastic tubes and external and internal lesions on the longitudinal section of the stems were measured.
Vol.: (0123456789) Fungal re-isolations were made from the lesion edges, to demonstrate Koch's postulates.Differences in lesion lengths between inoculum levels and species were analysed applying General Linear Model Univariate Analysis in IBM SPSS Statistics for Windows (Version 27.0).A similar experiment was conducted with green shoots in spring and assessed after 4 weeks.Assessments and data analysis were as described above for the one-year-old stems.

Effect of wounds on disease development
The effect of wounds on disease development by mycelia and conidia of D. seriata and N. parvum was studied using a detached stem assay on one-year-old stems and green shoots.This was followed by an experiment on disease development in glasshouse plants.

Detached stem assay
Asymptomatic one-year-old stems were surface sterilised as previously described.The most virulent isolates of D. seriata and N. parvum (DS27 and NP18), determined from the previous experiments were used for the mycelial inoculations.For the inoculation of wounded tissues, the stems were wounded in the midsection, inoculated with mycelial plugs, and wrapped with Parafilm ® while the mycelial plugs were placed directly onto the stem surface for non-wounded tissues.The control stems were inoculated the same way with PDA only.For the conidial inoculation, mixed-isolate conidial suspensions (10 4 conidia/mL) were prepared for the two species and inoculations were performed with a 50 μL of conidial suspension as described previously.The control stems were inoculated with SDW in the same way.The inoculated stems were placed in plastic tubes filled with SDW, arranged in a RCBD with 10 replicates, exposed to high humidity for 24 h, and maintained in a glasshouse for 6 weeks as in previous experiments.An identical experiment was conducted with green shoots in spring and assessed after 4 weeks.

Glasshouse plant assay
Asymptomatic potted plants of walnut, grown in the glasshouse for 20 months, were subjected to the same treatment combinations as for the detached stems.Each combination was assigned to five replicate plants in a RCBD.There were two inoculation sites in each plant in the middle of two green shoots (6-8 mm diameter) emerging from the main trunk.Following inoculation, the treated shoot was covered with a transparent plastic bag for 24 h and maintained in a glasshouse at ambient temperature of 17-25 °C.After 2 months, lesion assessments and fungal re-isolations were done.Due to the limited availability of plants, only exploratory shoots were observed for a longer period of 3 months.

Effect of wound age on disease progression
The susceptibility period of wounds to infection by D. seriata and N. parvum was studied using a detached stem assay, by exposing the wounds to mixed-isolate conidial suspensions of the species on days 0, 3, 7 and 14 post-wounding.An experiment with glasshouse plants was conducted, to study the susceptibility period of wounds for up to 4 months.

Detached stem assay
One-year-old stems were surface sterilised as previously described.Each stem was pruned by cutting the apical tip and pruning wounds were drop-inoculated on either Day 0, 3, 7 or 14 with 50 μL of mixed-isolate conidial suspension (10 4 conidia/mL) of either D. seriata or N. parvum.A non-inoculated control with SDW was included for each inoculation time.The trial was established with six replicates, and five stems allotted to each treatment within a replicate.Following inoculation on Day 0, the stems were placed in plastic tubes in SDW and arranged in a RCBD.For the latter inoculations, the pruned tip was moistened with a spray of SDW before inoculation.The stems were maintained in a glasshouse at ambient temperature of 17-25 °C for 6 weeks.For assessment, lesion lengths were measured as described in previous experiments.Fungal re-isolations were performed along the lesion edge, spanning the interface between normal and discoloured wood tissue.The presence or absence of mycelial growth characteristic of the inoculated species was recorded to determine mean percent recovery (MPR) of the pathogen, and MPR was analysed by Binomial Logistic Regression under Generalised Linear Models, in IBM SPSS Statistics for Windows (Version 27.0).

Glasshouse plant assay
Asymptomatic walnut plants grown in the glasshouse for 6 months were used for the experiment.For each plant, two one-year-old stems of similar diameter were tagged and randomly assigned to a treatment at four replicates plants per treatment.Each stem was pruned by cutting the apical tip, and pruning wounds were drop-inoculated on either Day 0, 3, 7, 14, 30, 60 or 120 with conidial suspensions of either D. seriata or N. parvum as described in the detached stem assay.Following inoculation, the plants were maintained in a glasshouse at ambient temperature of 17-25 °C.After 12 months, lesion lengths and data on MPR were recorded and analysed as described above in the detached stem assay.

Pathogen progression over time on glasshouse plants
Progression of D. seriata and N. parvum in glasshouse plants was observed over four time periods: 60, 120, 180 and 240 days.Asymptomatic walnut plants grown in the glasshouse for 20 months, were subjected to three inoculation treatments: conidia of D. seriata, conidia of N. parvum and negative control with SDW.The experiment was arranged in a RCBD with 12 combinations of treatment-assessment time.Each combination was allotted four replicate plants, with three inoculation sites in each plant.Two mixedisolate conidial suspensions (10 4 /mL) were made for N. parvum and D. seriata, as described above.In each plant, three green shoots (~6-8 mm diameter) emerging from the main stem were tagged and wounded at 15 cm from their bases with a sterile scalpel.Each wound was immediately inoculated with a 50 μL drop of the conidial suspension (10 4 /mL) and control plants were inoculated with SDW.The treated shoot was covered with a transparent plastic bag for 24 h.At 60, 120, 180 and 240 days after inoculations, lesion lengths and pathogen colonisation distances were assessed as in previous experiments.Pathogen re-isolations were performed from the lesion edges to confirm Koch's postulates.Lesion lengths and pathogen colonisation distances were analysed using General Linear Model Univariate Analysis as described above.

Symptom development
Twelve months post-inoculation, removing the bark revealed the healing of the inoculation wound in all the trunks (Fig. 1a).No external lesions were observed in 35 plants (83%).However, when the trunks were sectioned longitudinally in half, through the point of inoculation, internal discolouration of the wood was visible (Fig. 1b).Small external lesions were observed in 7 plants (17%) and the internal discolouration in those plants was much larger than the external lesion (Fig. 1c & d).The internal discolouration caused by N. parvum was dark in colour while the discolouration associated with D. seriata inoculations was lighter in colour (Fig. 1d & e).After winter dormancy, when the spring growth became visible, the buds close to the point of inoculation showed varied levels of dieback.The average number of buds with necrosis for the plants inoculated with N. parvum was four compared to one for D. seriata, and less than one for SDW.

Pathogen progression acropetally and basipetally
Lesion lengths above and below the point of inoculation indicated that the pathogen progressed more rapidly acropetally in the trunk than basipetally.While the mean acropetal movement of the six isolates was 28.4 mm (standard error of the mean SEM=2.7), the mean basipetal movement was 20.5 mm (SEM=2), which were significantly different (t=10.21,DF=35, P<.001).Correlation between the two measures was high with correlation coefficient of .95 and .96for D. seriata and N. parvum respectively.For the six isolates combined, the correlation between the two measures was .99,indicating that in almost all cases the pathogen moved more rapidly acropetally than basipetally.

Lesion development
All the inoculated pathogens produced lesions that were significantly longer than the control treatment and varied significantly in the lesion lengths they produced (F=282.4,DF=6, P<.001).Inter-species variation was significant with N. parvum producing lesions longer than that of D. seriata by 51.33 mm (SEM=1.5,P<.001).The longest lesions were produced by isolate NP18, followed by NP05 and NP03 (Fig. 2); intra-species variation among these three isolates was significant (F=32, DF=2, P<.001).Among the three D. seriata isolates, the intra-species variation was significant, with DS27 causing the longest lesions followed by DS04 and DS15 (F=4.84,DF=2, P=.024) (Fig. 2).

Pathogen progression beyond lesion edges
In all fungal inoculations, the inoculated pathogen was recovered beyond lesion edges.No pathogens were re-isolated from control treatments.For the three N. parvum isolates, the mean distance of isolation beyond the lesion edges varied from 22 to 36.3 mm (Fig. 2); intra-species variation among these three isolates was significant (F=97.5,DF=2, P<.001).Re-isolation of D. seriata beyond the margins of the lesion was smaller (7.8-9.8 mm) than that recorded for N. parvum (Fig. 2), and the intra-species variation was significant (F=9.3,DF=2, P=.002).
The paired sample t-test applied on lesion lengths and maximum re-isolation distances as measures of pathogenicity indicated that there was significant difference between the two measures (t=9.4,DF=35, P<.001).Correlation between the two measures was high with a correlation coefficient (r) of .85 and .92for D. seriata and N. parvum respectively, suggesting that isolates that caused longer lesions moved greater distance beyond the lesion edge.
To determine progression of the pathogen into the side shoots, the closest shoot to the inoculation point that was alive was assessed for discolouration.In the plants inoculated with N. parvum, a slight internal discolouration was observed in the side shoot (Fig. 1e).In plants where there was a co-dominance between the trunk and the side shoot, the internal discolouration in the side shoot was as dark as the internal lesion in the main trunk (Fig. 1f).In both cases, the pathogen was re-isolated from the discoloured wood, suggesting pathogen progression into the shoots.Isolations made at 10 mm intervals from the base of these shoots showed that N. parvum had progressed up to 20-30 mm in all the plants but had not reached the 40 mm mark in 12 months.In the plants inoculated with D. seriata, there was no visible discolouration at the base of the shoots closest to the

Infection potential of mycelia and conidia
On wounded one-year-old detached stems inoculated with either mycelia or conidia, the pathogen was reisolated from all inoculations.The control stems did not yield any fungal colonies typical of D. seriata or N. parvum.The lesion lengths caused by the six isolates were significantly different (F=567.54for mycelial and 248.26 for conidial inoculations, DF=6, P<.001) and had a similar ranking to the previous experiments; N. parvum causing significantly longer lesions than D. seriata for both mycelial and conidial inoculations (P<.001).The mean lesion lengths were in the range of 62-82 mm and 20-27 mm for mycelial inoculations of N. parvum and D. seriata, respectively (Fig. 3).For the conidial inoculations, the lesion lengths were in the range of 51-69 mm and 15-18 mm for N. parvum and D. seriata, respectively (Fig. 3).
Intra-species variation was also significant, with isolate NP18 causing the longest lesions followed by NP05 and NP03 in both type of inocula (Fig. 3).This is the same order that was recorded for the in planta assay at 1 year post-inoculation.Among the three D. seriata isolates, intra-species variation was observed only in mycelial inoculation, DS27 causing significantly longer lesions than DS15 (P=.008), and DS04 did not differ significantly from the other two.However, the three isolates did not differ significantly in lesion lengths they caused in conidial inoculation.
For all the six isolates, mean lesion lengths of mycelial inoculations were greater than those caused by conidial inoculations (Fig. 3).Analysis of the lesion lengths applying a paired sample t-test indicated that the difference in the two measures was significant, with a high correlation of .85 between the two measures (t=5.8,DF=35, P<.001).On green shoots, a similar pattern was observed for both mycelial and conidial inoculations, with a high correlation coefficient of .95 between the two measures (t=9.5, DF=35, P<.001).

Effect of inoculum concentration on disease incidence
All the conidial inoculations on one-year-old stems caused lesions irrespective of the concentrations (Fig 4a).Pathogen recovery was 100% for all concentrations of N. parvum (2-5000 conidia).For D. seriata, while inoculations with 50-5000 conidia resulted in 100% pathogen recovery, for the inoculations with concentrations, at P<.001.However, the lesion lengths of the two lowest concentrations of D. seriata with two to five conidia did not differ significantly from the dieback caused from the pruning tip in the control stems (P=.612 for ~five conidia).For N. parvum, the greatest mean lesion length of 84.8 mm (SEM=1.5)was recorded for 200 conidia which was significantly greater than the lesions produced with the lower concentrations (Fig. 4a).The higher concentrations resulted in decreased lesion length, but the lesion lengths did not differ significantly.For D. seriata, the longest lesion on one-year-old stems was caused by the application of 500 conidia (Fig. 4a).The lower concentrations produced significantly smaller lesions while the higher concentration did not result in any significant increase in lesion length.
On wounded green shoots, N. parvum exhibited the same pattern as in one-year-old stems with all the concentrations causing lesions that were significantly longer than the control at P<.001, with 200 conidia causing the longest lesions (Fig. 4b).For D. seriata, all inoculum levels produced lesions that were significantly longer than that of the control shoots, at P=.006 for 2 conidia and <.001 for all other concentrations.The longest lesion was caused by the application of 200 conidia which is less than the optimum inoculum level on one-year-old stems.For both species, the lower concentrations produced significantly smaller lesions while the higher concentrations did not result in any significant increase in lesion length.

Effect of wounds on disease development
Fungal re-isolations for D. seriata and N. parvum, inoculum types and tissue types are summarised in Table 1.On wounded detached one-year-old stems and green shoots, both mycelial and conidial inoculations of D. seriata and N. parvum caused lesions of varying lengths, and the inoculated pathogens were re-isolated from all lesion margins.However, on nonwounded detached stems and green shoots, conidial inoculation did not result in visible external or internal lesions.The mycelial inoculation caused a slight discolouration which became indistinct when washed in sterile water.The data on fungal re-isolation revealed that, on non-wounded one-year-old detached stems, mycelial and conidial inoculations of N. parvum and D. seriata resulted in low pathogen recovery from the bark (10-30%).While N. parvum was recovered from the wood after removing the bark, D. seriata was not recovered from the wood.Pathogen recovery from the bark was higher for both species and inoculum types than for the wood without the bark.Pathogen recovery for N. parvum was higher than D. seriata for both inoculum and tissue types.
On non-wounded green shoots of glasshouse plants, 2 months after inoculation, discolouration on the surface around the point of inoculation was visible for N. parvum which was more extensive for mycelial inoculation than the conidial inoculation (Fig. 5a, b &  c).The discolouration caused by D. seriata was light and insignificant.On peeling the epidermis (dermal tissue), the inner side of the tissue with mycelial inoculation had a light discolouration (Fig. 5d).
Pathogen re-isolation from the dermal tissue was 100% for both inoculation types and species (Table 1).For the inner tissues, pathogen recovery reduced to 70-80% and 20-30% respectively for N. parvum and D. seriata.For mycelial inoculation, pathogen re-isolation was slightly higher than that of the conidial inoculation.On both detached and attached tissues, the difference in disease incidence between the inoculations on wounded tissues and non-wounded tissues was so evident that no statistical analyses were performed.

Effect of wound age on disease progression
On one-year-old stems, wound age at the time of inoculation significantly affected infection by both D. seriata and N. parvum (P<.05).Mean infection incidence was 100% for wounds inoculated immediately after wounding on Day 0, for both species.For wounds inoculated on day 1 post-wounding, incidence of infection by N. parvum was 100% while it reduced to 93% for D. seriata.The incidence of infection for N. parvum reduced further to 83, 47, and 27% for 3, 7, and 14 day-old wounds, respectively (Fig. 6a).
A similar pattern was observed for D. seriata, but at a greater rate of reduction, with 67, 27 and 10% pathogen recovery for 3, 7, and 14 day-old wounds, respectively (Fig. 6b).While pathogen recovery for the day 0 inoculations were 100% for both species, for latter inoculations, pathogen recovery for N. parvum was consistently higher than D. seriata.Isolations from wounds on control stems did not yield any colonies characteristic of N. parvum or D. seriata.Mean lesion lengths caused by the two species also reduced significantly for the latter inoculations (P<.05).
On one-year-old stems in glasshouse plants, wound age at the time of inoculation significantly affected infection by both D. seriata and N. parvum, at P<.05.The mean incidence of infection for both species was 100% for wounds inoculated immediately after wounding (Fig. 7a).For wounds inoculated on day 3 post-wounding, incidence of infection by N. parvum was still 100% while it reduced to 88% for D. seriata.The incidence of infection for N. parvum reduced to 88, and 43% for 7, and 17 day-old wounds, respectively, and the lesion lengths were significantly different from each other and from day 0 and day 3 post-wounding inoculations (Fig. 7a).
A similar pattern was observed for D. seriata, but at a greater rate of reduction, with 55 and 25% pathogen recovery for 7, and 17 day-old wounds, respectively (Fig. 7b).The recovery of N. parvum for 30, 60, and 120 day-old wounds was in the range of 13-25% and they did not differ significantly from each other.Pathogen recovery for D. seriata from the 30 day-old wound was 13% and the lesion length did not differ significantly from the 17 day-old wound (Fig. 7b).Isolations from wounds on control plants did not yield any colonies characteristic of N. parvum or D. seriata.

Pathogen progression over time
Lesion assessment at 60 days after inoculation revealed significant differences between D. seriata and N. parvum with mean lesion lengths of 8.7 (SEM=1.5)and 21.2 (SEM=1.5)mm, respectively.With the progression of the lesions over time, mean lesion lengths caused by N. parvum were consistently greater than D. seriata (Table 2).Lesion lengths significantly progressed at 120, 180 and 240 days (F=168.63,DF=3, P<.001).Pathogen colonisation distance at 60 days, determined through re-isolation at 10 mm intervals, also showed significant differences between species with mean colonisation lengths of 19.17 (SEM=2.2) and 44.17 (SEM=2.2) mm for D. seriata and N. parvum, respectively, which were greater than the corresponding mean lesion lengths.Assessments at 120, 180 and 240 days post-inoculation indicated that the pathogen progression beyond the lesion progressed significantly over time for all the isolates (F=142.38,DF=3, P<.001) with N. parvum isolates showing significantly higher colonisation distances than the D. seriata isolates (Table 2).As the experiment progressed, the differences in pathogen colonisation between D. seriata and N. parvum also increased over time.

Discussion
This is the first study to confirm the pathogenicity of Botryosphaeriaceae isolates recovered from walnut orchards in Australia through an in planta assay.Although the detached stem assay by Antony et al. (2023) provided a preliminary indication of the pathogenicity of the species recovered from the walnut orchards in Australia, it should be noted that differences have been reported between the results of attached and detached assays in other crops (Amponsah, 2010;Scheper et al., 2018).Further, most species of Botryosphaeriaceae have a latent phase (Slippers & Wingfield, 2007), and development of disease symptoms is dependent on host-pathogen-environment interactions.Against this background, this research studied selected factors related to host-pathogen interactions that affect disease incidence and progression by Botryosphaeriaceae species in walnuts such as pathogen colonisation beyond dieback, effect of inoculum concentration on the severity of infection, effect of wounds on disease incidence and susceptibility period of wounds to infection.
In this study, differences were observed in dieback symptoms and the rate of symptom development between detached walnut stem and in planta assays.The mean lesion length caused by N. parvum during the 4 to 6 week incubation period in various detached stem assays was 62-85 mm.Similar sized lesions were not recorded in the glasshouse plants until one year post-inoculation.Wound was also observed on all the inoculation sites on the trunks of glasshouse plants one year post-inoculation.In 83% of the inoculated plants, wounds had healed to the extent that there was no visible external lesion, whereas none of the stems in the detached stem assay had any visible sign of wound healing when the experiment was concluded.Six weeks may be too short a period for visual observation of wound healing in detached stems.While the rate of symptom development varied between attached and detached assays in this study, the ranking of isolate aggressiveness was the same; isolates that caused longer lesions on detached stems, also caused longer lesions on attached stems.
The detached stem and in planta assays of this study confirmed that N. parvum was more virulent than D. seriata on the various tissue types evaluated (wounded, non-wounded, one-year-old stems, green shoots, attached tissues, detached tissues).On wounded tissues, N. parvum produced lesions that were approximately three times greater than those produced by D. seriata.This supports the findings of other studies originating from other geographical regions where N. parvum was found to be one of the most aggressive pathogens causing cankers and dieback in walnuts (Chen et al., 2014, Yu et al., 2015, López-Moral et al., 2020b, Gusella et al., 2021, Kara et al., 2021, Luna et al., 2022).Similar findings have been reported in other crops worldwide including grapevines (Billones-Baaijens, 2011), pistachio (López-Moral et al., 2020a) and almonds (Holland et al., 2021).Although D. seriata was found to be not as virulent as N. parvum, its prevalence and wide distribution in walnut orchards in Australia (Antony et al., 2023), and its ability to cause lesions that were significantly longer than the control treatments on all types of tissue tested, suggest that it should continue to be a pathogen of concern for Australian walnut production and should be investigated further.
In addition to inter-species variation, this study identified significant variation in pathogenicity among isolates of the same species.One explanation for this variation may lie in the amount of toxins the individual isolates can produce.Billones-Baaijens (2011) hypothesised that the genes that encode for toxin production may not necessarily be equally expressed in all isolates resulting in variability in their virulence.This is an area for further research in walnuts.
This study confirmed pathogen progression beyond the lesion edge to colonise the wood and found a strong linear relationship between lesion lengths and pathogen colonisation distances, with longer lesions indicating greater pathogen progression beyond lesion edges.This is not unexpected of necrotrophic pathogens.Among the three N. parvum isolates, NP18 was re-isolated the greatest distance beyond the lesion, followed by NP05 and then NP03.The same order was reflected in the lesion lengths the three isolates produced.A similar pattern was observed among the three D. seriata isolates.Therefore, it is reasonable to conclude that using any one of the two measures is adequate for pathogenicity analysis.Since determining pathogen colonisation distance is more time and resource consuming, lesion length as a simpler measure is acceptable for purposes of preliminary analyses.However, progression of the pathogens beyond the lesion has implications for the cultural practices adopted in the orchard for removal of stems and branches with dieback symptoms, which is essential to reduce inoculum load in the orchard.In this study, N. parvum recorded the longest colonisation distances of 36-44 mm beyond the lesion edge, depending on the type of tissue, type of inoculum and period of incubation.Therefore, when dead wood is removed from the orchard trees, to ensure the complete removal of infected wood, at least two more inches of the wood should be removed beyond the lesion edge.Similar recommendations have emerged from other studies in walnuts and pistachio (Michailides et al., 2016), in grapevine (Sosnowski et al., 2017), and in temperate zone nut crops (Moral et al., 2019).
The pathogenicity study on glasshouse plants showed that, as the pathogens moved acropetally and basipetally in the trunks, they killed the buds that would normally shoot following dormancy, with N. parvum killing more buds than D. seriata.Progression of N. parvum into side shoots was also greater compared to D. seriata.Studies have reported that N. parvum colonises the woody tissue and kills spurs and adjacent shoots, and dormant buds in grapevines (Amponsah, 2010;Úrbez-Torres & Gubler, 2011).In walnuts, the spread of species of Botryosphaeriaceae from the infected fruit to the peduncle and subsequently the spurs, killing the buds has been reported (Adaskaveg et al., 2017).As in the case of virulence, greater ability to colonise side shoots and buds may be attributed to the ability of N. parvum to produce various phytotoxins and enzymes to invade the plant cells.
The results presented here demonstrate that the isolates of N. parvum and D. seriata were able to infect non-wounded tissues, which is in agreement with Slippers and Wingfield (2007), who observed that Botryosphaeriaceae can infect through natural openings of plants.On pistachio, Michailides (1991) found that pycnidiospores of B. dothidea can infect uninjured fruit, leaves and stems through lenticels, stomata and scars created by detachment of petioles, buds or scale, by directly penetrating the plant tissues through the germ tubes.In a similar finding, Pusey and Bertrand (1993), while investigating the infection of peach by B. dothidea, reported that the pathogen invaded the non-wounded bark through lenticels and caused bark necrosis.On apple, conidia deposited on the fruit surface produced germ tubes that entered through lenticels or natural surface cracks (Kim et al., 1999).Where no lenticels or surface cracks were found, appressoria were frequently formed that penetrated the fruits.While the present study found that D. seriata and N. parvum were able to invade the non-wounded bark tissues and move to the wood below the bark, there were no lesions on the wood.This implies that the two species have moved into the wood and caused latent infection.This aligns with the findings of Tennakoon et al. (2017) who demonstrated that N. australe and N. ribis were able to latently infect non-wounded shoots of blueberry and progress endophytically.When and under what conditions they might change their latent phase to become pathogenic is unclear.Several studies have attributed this change to environmental factors, plant stress and competition for resources among the endophytic microbial community (Slippers & Wingfield, 2007;Sakalidis et al., 2011).However, after a review of related research, Manawasinghe et al. (2016) argue that it is the increased host susceptibility that triggers this change and that environmental factors may not have a direct effect in triggering pathogenicity in the Botryosphaeriaceae.Further research is needed to understand the change from the latent phase to the pathogenic phase of Botryosphaeriaceae species in walnuts.
The experiment on inoculum potential revealed that exposure of the wound to even two conidia of N. parvum, and between five and 20 conidia of D. seriata, caused significant disease incidence on detached one-year-old stems, under glasshouse conditions.This endorses the findings of Amponsah (2010) and Sosnowski et al. (2017) with N. luteum inoculum on detached canes of grapevines.In the absence of plant defence mechanisms, the glasshouse conditions might have provided optimum conditions for infection of detached stems by such a low inoculum level.Occurrence of such conditions in the field cannot be underestimated and therefore, even in orchards with low disease incidence, removing the inoculum sources such as infected wood from the orchard is essential.A cultural practice that is often underestimated for its ability to spread low levels of inoculum is the orchard equipment.Studies have found that pruning tools and orchard equipment can carry inoculum and spread infection by pathogenic fungi and bacteria (Gramaje & Armengol, 2011;Billones-Baaijens et al., 2013;Agustí-Brisach et al., 2015;Nguyen et al., 2017).Adopting appropriate sanitisation protocols between and during use in the orchard, even if the orchard has low disease incidence, will be necessary to reduce further spread of the infection.
With increase in inoculum level, this study found that the lesion lengths initially increased significantly, and then plateaued for both species and tissue types.On green shoots, greatest lesion lengths were recorded for 200 conidia and exposure to higher inoculum levels (up to 5000 conidia) did not result in further increase in lesion length.This result confirms the findings of Sosnowski et al. (2017) who reported that, under greenhouse conditions, 200 to 1000 N. luteum conidia have similar infection potential on one-yearold detached canes of grapevines.However, this is contrary to studies that have reported increasing disease severity with increasing inoculum concentration.Pusey and Bertrand (1993) found that increasing concentrations of B. dothidea inoculum resulted in a greater area of necrosis on non-wounded bark of peach trees.There have also been studies that showed that, after an initial trend of increasing disease severity with increase in inoculum concentration up to a point, for higher concentrations the disease severity reduced significantly.On detached canes of grapevines, Amponsah (2010) recorded longer lesions with 10 4 conidial/mL than with 10 6 and 10 5 conidial/mL and hypothesised that the reduction in disease severity may be due to competition for resources on the small inoculation site.
The broad range of inoculum level that can cause significant infection implies that, irrespective of the inoculum load, orchard practices that might cause injuries to the plants should be avoided when the field conditions are conducive for inoculum release and dispersal.While various factors such as humidity, exposure to sunlight, radiation and temperature can affect conidial survival, rain events have been found to be highly correlated with conidial release (Úrbez-Torres et al., 2010;Billones-Baaijens et al., 2017).Since rain events facilitate both conidial release and transmission to wounded plant tissues through watersplash, the best cultural practice to reduce infection incidence is to schedule pruning for dry periods.In walnut orchards, the window for pruning begins in autumn after harvest and ends by mid-winter.There have been contradictory research outcomes on the effectiveness of autumn pruning vs winter pruning in various geographical regions and the rainfall pattern of those locations might partially explain the contradiction (Michailides et al., 2016;Moral et al., 2019; Vol.: (0123456789) Billones-Baaijens & Savocchia, 2019).In Australia, the major walnut growing areas have very different rainfall patterns, some with winter dominant rains and some with the lowest rainfall in winter.The pruning period for these regions needs to be identified after considering the rain forecast for the respective locations.
While identifying a suitable window for pruning, it is important to consider the effect of pruning wounds on infection spread and how long the wounds are susceptible to infection.Long lesions, visible symptoms of necrosis, and pathogen colonisation beyond the necrosis, recorded in this study on inoculation of wounded tissues, clearly indicate that wounded tissues are primary sites of infection for N. parvum and D. seriata.This aligns with Brown and Hendrix (1981) who observed that, conidial germ tubes consistently grew towards injured tissue.Further, the experiments on wound age revealed that pruning wounds were highly susceptible to infection during the first week after pruning, when the infection incidence was in the range of 88-100% for N. parvum and 55-100% for D. seriata; infection incidence reduced significantly for the latter inoculations.Reduction in the susceptibility of wounds to infection over time has been reported in almonds (Holland et al., 2023) and grapevines (Amponsah, 2010;Úrbez-Torres & Gubler, 2011;Sosnowski et al., 2023).However, this contradicts the results of field trials from California walnut orchards where susceptibility of pruning wounds to infection did not decrease even 4 weeks after pruning (Michailides et al., 2014).They found that the length of cankers caused by the latter inoculations was similar to those caused by inoculations done immediately after pruning.The trial reported in 2017 also found that the pruning wounds were highly susceptible even after 120 days of wounding (Michailides et al., 2017).This finding was interpreted as an indication that walnut wounds take a long time to heal.The researchers also hypothesised that pruning cuts that create a wound at the tip of the stems may have an extended period of susceptibility due to the nature of the loosely packed pith that creates an open channel in the middle of the stems.When fungal inoculum lands in this channel, it may be absorbed by the pith and initiate infection internally.In field trials, the role of inoculum that is already present in the orchards, and consequent early natural infection as soon as wounds are made cannot be ruled out.However, the in planta assay conducted in the controlled environment of the glasshouse, as part of this study, minimised the possibilities of background infection and showed that wound susceptibility decreased significantly with wound age; the highest infection occurring in the first week.Therefore, in addition to reducing inoculum levels in the orchard, it is critical to protect the wounds during the first week following pruning.
While wound healing was observed on the trunks of the glasshouse plants, a similar development was not visible with the pruning wounds at the tip of the stems, although both experiments had the same glasshouse conditions and the same incubation period of 12 months.Biggs (1987) examined responses to mechanical wounding of 17 tree species and reported that, in most species, continuous suberised boundaries developed following wounding, forming impenetrable layers in the xylem vessels.In a similar study, Woodward and Pocock (1996) examined the development of wound periderm after mechanical wounding on the barks of four tree species (Acer pseudoplatanus, Betula pendula, Fagus sylvatica and Sorbus aucuparia) and detected the ligno-suberized barrier zone within 7 days.They found that cell walls had thickened extensively in 28 days and the wound periderm was fully formed.These studies have assessed the wound responses of the bark tissues; a similar histological and histochemical study of pruning wounds caused at the tip of the stems and branches, may shed light on protecting the walnut trees during and after pruning.
This study has provided an understanding of some of the key aspects of pathogenicity and progression of infection of walnut by N. parvum and D. seriata in walnut orchards in Australia.It highlights orchard management practices that need to be implemented to reduce inoculum availability, inoculum dispersal and injuries to walnut trees.Removal of infected wood from the orchard, avoiding pruning under wet conditions, protecting wounds during the first week following wounding, and following orchard hygiene are some of the management options to reduce disease incidence.Areas of research that warrant further investigation are also highlighted.

Fig. 1
Fig. 1 Typical symptoms on trunks of glasshouse plants 12 months post-inoculation of Diplodia seriata and Neofusicoccum parvum (a) Wound healing at the site of inoculation (blue arrow); (b) internal lesion caused by N. parvum under the healed inoculation wound; (c) small external lesion at the site of inoculation observed in some plants (red arrow); (d) inter-

Fig. 2
Fig. 2 Pathogenicity of isolates of Neofusicoccum parvum (NP03, NP05, and NP18) and Diplodia seriata (DS04, DS15, and DS27) as measured by the mean lesion lengths (a-f) and maximum pathogen re-isolation distances (A-E).The error bar represents the standard error of the mean.Different letters on

Fig. 3
Fig. 3 Mean lesion lengths caused by mycelial and conidial inoculations of Neofusicoccum parvum (NP18, NP05, and NP03) and Diplodia seriata (DS27, DS04, and DS15) on wounded one-year-old detached stems.The error bar represents the standard error of the mean.Different letters on the

Fig. 5
Fig. 5 Effect of mycelial and conidial inoculations on nonwounded green shoots of glasshouse plants (a) cup like structure for conidial inoculation of non-wounded shoot; (b) discolouration on the external dermal tissue caused by conidial

Table 1
Pathogen re-isolation from non-wounded inoculations of different tissue types

Table 2
Pathogen progression of Neofusicoccum parvum and Diplodia seriata over four post-inoculation times Values followed by different letters are significantly different according to Tukey's HSD at P<.05.Means of species (a-b & p-q) were significantly different (P<.001).Means for months of assessment (c-f & r-u) were significantly different (P<.001)