Infection Of Grapevine Tissue By Botryosphaeria Species

Infection of grapevine tissue by Botryosphaeria species Nicholas Amponsah, Eirian Jones, Hayley Ridgway and Marlene Jaspers Bio-Protection and Ecology Division, Lincoln University, PO Box 84 Lincoln University, Canterbury, New Zealand Abstract The Botryosphaeria species isolated from symptomatic grapevines, B. lutea (Neofusicocum luteum), B. australis (Neofusicocum australe) B. parva (Neofusicocum parvum) B. stevensii (Diplodia mutila) and Botryosphaeria sp. were inoculated onto to wounded green shoots of grapevines on which they were shown to be pathogenic. The brown lesions that developed were able to exude numerous conidia for all Botryosphaeria species tested except B. parva. Non-grapevine isolates of B. australis, B. stevensii and a Botryosphaeria sp. were also found to be equally pathogenic on grapevines. The shoots of the grapevine cultivars, Chardonnay, Pinot noir, Riesling, Cabernet sauvignon and Sauvignon blanc were also equally diseased by mycelium of B. lutea, B. parva and B. stevensii. When mature trunks of 18 month Pinot noir plants were inoculated with B. lutea conidia and mycelium the plants showed no visible symptoms, however the pathogen was found to have progressed through the plant to the pruned tip, from where it then caused dieback. This research has shown that the Botryosphaeria species from infected grapevines and other woody hosts are able to produce symptomatic infections on green grapevine shoots of the commonly-grown varieties. However on woody shoots, the infections are initially endophytic and symptomless, progressing quickly through the shoots to pruning wounds from which they cause dieback. 1 Introduction Pathogenic Botryosphaeria species cause economic losses world-wide on a wide range of woody plant hosts of agricultural, horticultural and forestry significance. On grapevines, Botryosphaeria spp. are often found associated with dieback of trunks, pruned or trimmed shoots and canes, as well as incomplete graft unions, bud mortality and a general loss of vigour termed ‘grapevine decline’. Worldwide, at least 11 Botryosphaeria species have been identified as important grapevine pathogens, but species distribution and their pathogenicity appears to vary between countries, possibly due to climatic factors. Very little research has been conducted on the infection processes of the Botryosphaeria spp. in grapevines and the factors that enhance infection risk, knowledge that is essential to the development of control strategies. However, the reported research on Botryosphaeria diseases of other crop species, and observations in vineyards has provided some relevant information. In Botryosphaeria diseases of other crops, spores were shown to infect through pruning wounds, and to cause similar symptoms to those in grapevines. However, the reported grapevine infection studies have mostly used mycelial plugs applied to the wounds. Since it is more likely that spores such as conidia are the main inoculum source in vineyards, they should also be tested. This research reports on the preliminary infection studies that tested the infection potential of the Botryosphaeria species found within New Zealand against the main grape varieties grown here. To ensure that the research was robust and relevant, both conidium and mycelial inoculation methods were used. 2 Materials and Methods 2.1. Pathogenicity of common Botryosphaeria species from New Zealand vineyards Three isolates each of B. lutea (Neofusicocum luteum), B. australis (Neofusicocum australe) B. parva (Neofusicocum parvum), B. stevensii (Diplodia mutila), two isolates of B. obtusa (Diplodia seriata) and one isolate of a Botryosphaeria sp. were cultured on potato dextrose agar (PDA) and incubated at 24oC in continuous darkness for 3 days. Mycelium agar discs (3 mm) cut from the pathogen cultures were inoculated onto wounded sections of 20-25 cm

long green Pinot noir shoots cut from actively growing field vines. Controls were inoculated with sterile agar plugs. The base of each shoot was inserted into a Universal bottle containing sterile distilled water in an enclosed transparent chamber at room temperature (15-23°C), with frequent misting for the first 3 days. The assessments of lesion length were made on all plants at 10 days, with isolations made from three plants per treatment. The lesions on the three remaining plants from each isolate were surface sterilised, air dried and put in a moist container, to induce pycnidium formation and oozing of conidia (spores). Data of lesion lengths were analysed by one way ANOVA using GENSTAT 9.0.

A

B

Figure 1A. In vitro inoculation of a grapevine shoot with a mycelium colonised agar plug (a) 1B. Lesion length at 7 days on an inoculated plant (b), and no lesion (c) on a control plant. 2.2. Grapevine cultivar susceptibility to three Botryosphaeria species The shoot susceptibility of five grapevine cultivars: Chardonnay, Pinot noir, Riesling, Cabernet sauvignon and Sauvignon blanc was studied using mycelium colonised agar plugs of B. lutea, B. parva and B. stevensii isolates, one each, which were initially isolated from New Zealand grapevines. Prior to the inoculations, the green shoots were either wounded by cutting a 3 mm wide and 2 mm deep hole or non-wounded. The discs cut from the Botryosphaeria cultures were inserted mycelium side first into the wounds in the centres of the grapevine shoots (Fig. 1A), six shoots per isolate, which were incubated as described previously. For non-wounded treatments, the agar plugs were placed onto the shoots and held in place with cling film. Control plants were inoculated with sterile agar. 2.3. Pathogenicity of non-grapevine Botryosphaeria isolates on grapevine shoots The 10 isolates of Botryosphaeria species found in symptomatic non-grapevine hosts (Table 3) were inoculated onto green shoots of Pinot noir, as described above. There were six replicates per isolate with plants arranged in a completely randomised design. Assessment of lesion length was made on all six plants and three were used for pathogen isolation. The lesion areas from the remaining three plants per isolate were dried at room temperature, moistened and incubated to induce pycnidium and conidium production.

2.4. Susceptibility of mature canes to infection by B. lutea Potted Pinot noir grapevine plants (18 months old) were wounded by drilling 3.5 mm diameter holes into the wood and inoculated with Botryosphaeria lutea (the most pathogenic species) using mycelium agar discs (3 mm) or conidia (104/mL). The 10 replicate plants per treatment were arranged in a randomised complete block design within a shade house. Disease assessment was carried out monthly for 4 months. Due to difficulty seeing the lesions, pathogen position was determined by isolating from wood segments taken at 10 mm intervals from the inoculation point. 3. Results and Discussion 3.1. Pathogenicity of common Botryosphaeria species from New Zealand vineyards There were significant differences (P≤0.05) in the pathogenicity of the Botryosphaeria isolates and species, as shown by the mean lesion lengths (Table 1 and Fig 2). At 10 days after inoculation, the B. lutea isolates [M (13) 2, G-14 and N (12)2] had the longest lesions followed by isolate I-1 (Botryosphaeria sp.) and then B. australis isolates Mel-2, J-3 and Kat1. The next most pathogenic isolate Q-2 (B. parva) was followed by Iso-2 (B. stevensii), and both were significantly different (P≤0.05) from the other B. parva isolates [I (15)2 and I (15) 3] and B. stevensii isolates [F (12)2 and Q]. Similar differences in pathogenicity among isolates of the same species have been reported by many researchers. In contrast to many overseas reports of the B. obtusa, the isolates L-I and L (16)2 were not pathogenic to the grapevine shoots since the lesions extended little beyond the wounded inoculated points, being similar (P≥0.05) to the controls. Table 1. Mean lesion lengths caused by Botryosphaeria isolates on wounded green shoots of Pinot noir.

1

Species name

Isolate

B. lutea B. lutea B. lutea B. australis B. australis B. australis B. parva B. parva B. parva B. stevensii B. stevensii B. stevensii B. obtusa B. obtusa Botryosphaeria sp. Sterile agar Control LSD (P≤0.05)

N(12)2 M(13)2 G-14 Kat-1 J-3 Mel-2 Q-2 I(15)2 I(15)3 Iso-2 F(12)2 Q L(16)2 L-1 I-1

Mean lesion length (mm) 76.13 g1 71.12 fg 68.98 efg 65.93 defg 65.85 defg 62.66 cdefg 58.70 cdef 53.04 cd 48.53 c 55.42 cde 26.33 b 24.23 b 10.07 a 8.38 a 67.28 defg 0.00 a 13.36

Means followed by different letters are different (P≤0.05) by Fisher’s protected LSD test.

Figure 2. Shoot lesions at 10 days after inoculation with three isolates each of (A) B. lutea (B) B. australis (C) B. parva (D) B. stevensii and (E) B. obtusa.

3.2. Grapevine cultivar susceptibility to three Botryosphaeria species The non-wounded shoots of all cultivars had no significant lesions for all the Botryosphaeria isolates tested, being similar to that of the controls (data not included). However with the wounded inoculated shoots, symptoms of brown to dark brown shoot lesions (as in Fig 1B) were observed on all five cultivars. After 10 days, lesion lengths differed significantly (P≤0.05) among the pathogens (B. lutea, B. parva and B. stevensii) used to test for the cultivar susceptibility (Fig 3), and all caused shoot death by 2-3 weeks. However, there was no significant difference in lesion length of the five grapevine cultivars tested (P>0.05; Fig.4), indicating that they were equally susceptible to the Botryosphaeria species tested. The inoculated isolates were all re-isolated from the shoots together with a few other fungi.

Mean lesion lengths (mm)

30 25

C

20

B

15 10

A D

5 0 B. stevensii

B. parva

B. lutea

Control

Botryosphaeria species

Figure 3. Mean lesion lengths, indicating pathogenicity of B. lutea, B. parva and B. stevensii on five grapevine cultivars (Bars with the same letters do not differ significantly (P≤0.05) by Fisher’s protected LSD test).

Figure 4. Mean lesion lengths at 10 days after inoculation with mycelium plugs of B. lutea, B. parva and B. stevensii, indicating susceptibility of grapevine cultivars (bars with the same letters do not differ significantly (P≤0.05) by Fisher’s protected LSD test)

3.3. Pathogenicity of non-grapevine Botryosphaeria isolates All the Botryosphaeria isolates from the non-grapevine hosts were able to infect grapevine shoots, producing similarly long lesions to the grapevine isolates and differed significantly (P≤0.05) from the controls (Table 2). This indicates the potential of other hosts to provide inoculum sources for infection of vineyards. Pathogen re-isolation yielded 100% recovery of the inoculated isolates together with a few other fungi. All of the isolates were able to produce pycnidia with copious conidium ooze exuding from them after incubation of the infected shoots under moist conditions (Fig. 5). Table 2. Pathogenicity of Botryosphaeria isolates from non-grapevine. Species name

Isolates

Hosts

B. australis B. stevensii B. stevensii B. stevensii B. stevensii B. stevensii Botryosphaeria sp. Botryosphaeria sp. Botryosphaeria sp. Botryosphaeria sp. Control LSD (P≤0.05)

J-3 Iso-2 J-4 A-1 F-1 A-2 O-3-1 O-1 C-4 I-1 Sterile agar

Broom Native ngaio Oak Plum Willow Apple Cherries Pine Olive Lemon wood

1

Lesion length (mm) 68.4 f1 57.9 e 51.0 de 39.6 bc 40.7 bcd 34.0 b 46.1 cd 44.3 bcd 43.8 bcd 69.8 f 0.0 a 10.03

Means followed by different letters are different (P≤0.05) by Fisher’s protected LSD test.

Figure 5. Pycnidia oozing conidia of Botryosphaeria sp. after incubating lesion shoots under moist condition.

3.4. Susceptibility of mature canes to infection by B. lutea Pathogen position within the wood showed that after conidium inoculations, the pathogen progression was slower than with the mycelium inoculations. With both types of inoculation, the pathogen progressed through the tissue, moving faster in the upward than the downward direction (Fig. 6). By the 4th month the pathogen had entered the first side shoots just past the inoculation point, still progressing without any visual symptoms. Once the pathogen progressed to the tip of the shoot, symptoms of dieback began to appear at the pruned tip.

Distance moved by pathogen (mm)

100

Conidia ▲ Mycelium ▲

80

Conidia ▼ Mycelium ▼

60 40 20 0 -20 -40 Dec-07

Jan-08

Feb-08

Mar-08

Apr-08

Dates on which pathogen was assessed

Figure 6. Botryosphaeria lutea progression upwards and downwards from points of inoculation with conidia and mycelium discs. in 18 months old Pinot noir grapevine trunks. 4. Conclusion This research has shown that the Botryosphaeria species present in and around New Zealand vineyards were pathogenic to the commonly grown grapevine cultivars. They were able to produce numerous spores that could infect through wounds in green shoots, on which they caused severe lesions, and in the mature canes, where visible symptoms did not developed immediately at the infection points. In the mature wood, the pathogen progressed rapidly through the plant to the pruned tip from which it caused then dieback. This has clear implications for development of pruning strategies and the need to protect pruning wounds from potential infection. 5. Acknowledgements The authors gratefully acknowledge the many New Zealand grape growers who provided practical help for this project, and New Zealand Winegrowers and Lincoln University who provided funding.