Virulence and cross-infection potential of Ilyonectria spp. to grapevine

Headnote

Summary. Black foot is an important disease of grapevines, affecting vines in nurseries as well as in young plantations. In recent years the disease has increased in incidence and severity throughout the world. Black foot is associated with at least two Campylocarpon and 12 Ilyonectria species, most of which have only recently been described. The recognition of previously unknown species, together with published reports of variability in virulence between and within species identified as I. macrodidyma and I. liriodendri, underlined the need to compare the virulence of isolates from these complexes. A further objective of this work was to determine the cross-infection potential of isolates of these species from other hosts to grapevine. Results from this study revealed recently described species such as I. lusitanica, I. estremocensis and I. europaea to be more virulent to grapevine than the species previously accepted as the main causal agents of black foot, such as I. liriodendri and I. macrodidyma. Furthermore, these results also provided support for isolates obtained from non-grapevine hosts to be as virulent to grapevines as isolates obtained from grapevine, underlying the cross-infection potential of these pathogens.

Key words: black foot disease; Cylindrocarpon root rot; pathogenicity; Vitis vinifera.

Introduction

Black foot is an important disease of grapevines in most countries throughout the world. In recent years the disease has been reported with an increased incidence and severity, affecting both nurseries and young plantations, causing typical darkening of the basal end of rootstock plants (Halleen et al., 2004; Oliveira et al., 2004). Declining plants are frequently found in infected vineyards, showing slow growth, reduced vigour, retarded sprouting, shortened internodes, sparse and chlorotic foliage (Rego et al., 2000; Halleen et al., 2006a), resulting frequently in plant death and forcing growers to replant considerable areas.

Black foot is caused by several Cylindrocarpon-like species residing in the genera Campylocarpon and Ilyonectria. Two species of Campylocarpon have been reported, namely Campyl. fasciculare Schroers, Halleen & Crous and Campyl. pseudofasciculare Halleen, Schroers & Crous (Halleen et al., 2004), although these have thus far only been reported from South Africa (Halleen et al., 2004) and Uruguay (Abreo et al., 2010). The genus Ilyonectria was recently established within what was formerly regarded as Neonectria s. lat., accommodating well-known pathogens such as Ilyonectria liriodendri (Halleen, Rego & Crous) P. Chaverri & C. Salgado and I. macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado (Chaverri et al., 2011). In fact, I. liriodendri and I. macrodidyma are the species most commonly reported from affected grapevines (Petit and Gubler, 2005; Halleen et al., 2006b; Alaniz et al., 2007). Recent studies have shown, however, that many of these records actually represent some newly described species (Cabral et al., 2012a, 2012b). These include I. alcacerensis A. Cabral, Oliveira & Crous, I. estremocensis A. Cabral, Nascimento & Crous, I. novozelandica A. Cabral & Crous and I. torresensis A. Cabral, Rego & Crous which were described from within the I. macrodidyma species complex (Cabral et al., 2012b), and I. europeaea A. Cabral, Rego & Crous, I. lusitanica A. Cabral, Rego & Crous, I. pseudodestructans A. Cabral, Rego & Crous, I. robusta (A.A. Hildebr.) A. Cabral & Crous and I. vitis A. Cabral, Rego & Crous, which emerged from the I. radicicola (Gerlach & L. Nilsson) P. Chaverri & C. Salgado species complex (Cabral et al., 2012a). Ilyonectria torresensis was found to be associated with Vitis vinifera, Abies nordmanniana, Fragaria sp. and Quercus sp. in countries throughout the world. In contrast, I. alcacerensis has thus far only been reported from V. vinifera in the Iberian Peninsula. Ilyonectria novozelandica was associated with V. vinifera in New Zealand, South Africa and the USA, but also reported on Festuca duriuscula in Portugal. Ilyonectria estremocensis was isolated from V. vinifera in Portugal and Picea glauca in Canada (Cabral et al., 2012b). Ilyonectria europaea, I. pseudodestructans and I. robusta were found on V. vinifera in Portugal and on other host plants in different parts of the world, while I. lusitanica and I. vitis were thus far exclusively reported from grapevines (Cabral et al., 2012a). Besides these, "Cylindrocarpon" pauciseptatum Schroers & Crous was associated with diseased roots of Vitis spp. in New Zealand and Slovenia (Schroers et al., 2008), in Uruguay (Abreo et al., 2010), in Portugal (Cabral et al., 2012a) and in Spain (Martín et al., 2011).

Ilyonectria macrodidyma was reported as more virulent to grapevines than I. liriodendri, although variation in virulence among groups of I. macrodidyma was also found (Alaniz et al., 2009). However, no other comparative virulence studies have been reported among the pathogens causing black foot disease of grapevine. This is becoming particularly relevant, as at least 12 species are currently recognised to be associated with this disease. Moreover, most of these species are not exclusive to grapevine, and infect several other hosts, underlining the cross-infection potential of isolates from other hosts to grapevines. Therefore, the objective of this work was to compare the virulence of isolates from different species associated with black foot disease of grapevines, as well as to test the pathogenicity of isolates from other hosts to grapevine.

Materials and methods

A total of 60 isolates were analysed, 36 of which are from grapevines (Table 1). The other hosts include Olea europaea (five isolates) and Quercus spp. (five isolates), among others. Species covered in this study include "C." pauciseptatum (three isolates from grapevine and one from Olea europaea), I. alcacerensis (two isolates from grapevine), I. estremocensis (four isolates from grapevine), I. europaea (two isolates from grapevine, one from Actinidia chinensis and one from Aesculus hippocastanum), I. liriodendri (four isolates from grapevine, one from Liriodendron tulipifera, one from Malus domestica, and one from Quercus suber), I. lusitanica (one isolate from grapevine), I. macrodidyma (three isolates from grapevine and two from Olea europaea), I. novozelandica (five isolates from grapevine and one from Festuca duriuscula), I. pseudodestructans (two isolates from grapevine and two from Quercus sp.), I. robusta (three isolates from grapevine, two from Quercus spp., two from Panax quinquefolium, one from Prunus cerasus, one from Tilia petiolaris, one from Thymus sp. and one from an aquarium with Anodonta), I. vitis (one isolate from grapevine), I. torresensis (four isolates from grapevine, one from Fragaria x ananassa and one from Olea europaea), an I. estremocensis- like undescribed species, here referred as llyonectria sp2 (L. Mostert, personal communication; two isolates from grapevine, one from Pinus laricio and one from an unknown host) and an I. venezuelensislike undescribed species, here referred as llyonectria sp1 (Cabral et al., unpublished data).

Cuttings of the susceptible rootstock 1103P (Alaniz et al., 2010) were rooted for 1.5 to 2 months at 20°C in a rooting bench containing perlite and sand. Irrigation was carried out by overhead nebulisation for 5 s every 10 min.

After the rooting period, plants were removed from the bench and the roots were slightly pruned. The wounded cuttings were dip-inoculated by immersing the roots and the basal end of the cuttings in a 106 mL-1 conidial suspension (for each isolate listed in Table 1), for 60 min. Conidia were harvested by flooding 14 d old potato-dextrose agar (PDA, Difco, USA) cultures with sterile distilled water, and dislodged with a sterile glass rod. The spores and mycelium were then filtered through a double layer of cheesecloth, and the conidial concentration estimated using a haemocytometer, which was then adjusted with sterile distilled water. After inoculation, the rooted cuttings were planted in 1 L bags containing a mixture of soil, peat and sand (2:1:1, v/v/v), and maintained in a greenhouse at 24±5°C (day) and 18°C (night) with approximately a 12 h photoperiod. For negative control plants, sterile distilled water was used instead of conidial suspension.

The plants were grown on the greenhouse for 4.5 months and, following this period, results were evaluated for each isolate (10-12 plants per isolate, including the control), and compared to the control. The parameters analysed were focused on the loss of root (number and root dry weight, and the length of the longest root) and shoot (number of shoot nodes and the length and shoot dry weight; usually a single shoot was formed) biomass and on the intensity of wood colonisation by the pathogens (percentage of reisolation). For the latter, 10 pieces of wood from the basal end of each rootstock plant (at least 2 cm above the bottom) were excised, disinfected (for 1 min in a NaClO solution with 0.35% w/w as active chlorine), rinsed with distilled water and placed in Petri dishes containing PDA amended with 250 mg L-1 chloramphenicol (Bi°Chemica, AppiChem, Germany). The dishes were incubated at 20°C for up to 2 weeks and observed for the presence of Ilyonectria colonies, which was confirmed through morphological appearance of colonies and conidial characteristics. The percentage of reisolation was calculated as the proportion of wood pieces from which Ilyonectria colonies were recovered, versus the total number of pieces of wood for each plant.

All data were subjected to a one-way ANOVA and means compared using the Tukey's test at a 5% significance level (STATISTICA 8.0). Before analysis, arcsine-square root transformation was performed for data expressed as percentage.

To confirm results from this experiment, data from a subsequent, smaller experiment were used for comparison under the same conditions as stated above. Isolates tested were from the following species: I. estremocensis (isolates Cy135, Cy144, Cy145, Cy152 and Cy153 from grapevine), I. europaea (isolate Cy131 from Actinidia chinensis), I. liriodendri (isolates Cy5, Cy68 and Cy76 from grapevine, Cy164 from Malus domestica and Cy232 from Quercus suber), I. novozelandica (isolate Cy230 from Festuca sp.), I. pseudodestructans (isolates Cy20 and Cy22 from grapevine, and CBS 117812 from Quercus sp.), I. robusta (CBS 117818 from Quercus sp. and Cy231 from Thymus sp.), I. torresensis (isolates OL1 from Olea europaea, Cy96 from Quercus sp. and Cy221 and Cy222 from Fragaria x ananassa) and Ilyonectria sp1 (isolate OL2 from Olea europaea).

Results

At the end of the first experiment, root rot symptoms were visible in inoculated plants, in contrast to the uninoculated control plants. Symptoms included root lesions, vascular discolouration, and necrosis in the basal plant tissues, although the quantification of these lesions and discolouration was not possible. Symptoms related to reduced vigour were more readily quantifiable. In general, inoculated plants had shorter shoots with less nodes, as well as less and shorter roots, although significant differences were found among isolates (Table 2).

The percentage of reisolation ranged from a minimum of 18.6% for isolate OL2 (llyonectria sp1, from Olea europaea) to a maximum of 96.5% for isolate CBS 537.92 (I. europaea, from Aesculus hippocastanum). Control plants had 0% reisolation, differing significantly from all tested isolates except OL2 and Cy230. This trait had the fewest homogeneous groups among all the traits studied.

The average number of roots in the control plants was 36.3, which did not differ significantly from the maximum value recorded from inoculated plants (35.8 for isolate CBS 112615; I. macrodidyma from grapevine). The minimum value for NR was 19.2 for isolate CBS 117526 (I. liriodendri, from grapevine), which represents a 47% reduction in the number of roots.

The root dry weight ranged from a maximum of 4.50 g for isolate OL2 (which did not differ significantly from the control plants; 4.08 g) to a minimum of 0.49 for Cy243 (I. estremocensis, from grapevine; 88% reduction from control).

The length of the longest root for the control plants was 49.6 cm, with all inoculated plants showing a significant reduction from that value, ranging from a minimum reduction of 23% for isolate OL2 to a maximum of 66% (16.8 cm) for isolate Cy243.

The average number of shoot nodes in the control plants was 15.9, ranging for the inoculated plants from 13.2 nodes for isolate Cy233 (I. vitis, from grapevine), which did not differ significantly from the control, to 8.1 nodes for isolate CBS 129086 (I. torresensis, from grapevine), which represents a 49% reduction.

The average shoot length was 52.2 cm in the control plants, ranging from 43.0 cm for isolate Cy200 (llyonectria sp2, from grapevine; 43.0 cm, 18% reduction) to 18.1 cm for isolate Cy243 (18.1 cm, 65% reduction).

The shoot dry weight ranged from a maximum of 1.08 g for isolate CBS 113552 (I. novozelandica, from grapevine), which did not differ significantly from the control (0.95 g), to a minimum of 0.22 g (Cy243), which represents a 80% reduction.

Considering the isolates obtained from grapevine separate from the isolates from other hosts, significant differences were observed among species and to the control (Figure 1). The percentage of reisolation ranged between 39.4% for I. pseudodestructans and 85.0% for I. vitis for grapevine isolates, all of them differing significantly from the control. Results for isolates from other hosts ranged between 88.1% for I. torresensis and 18.6% for Ilyonectria sp1 (which did not differ from the control, along with I. novozelandica; the latter was the single species with significant differences among isolates from grapevine and other hosts).

For grapevine isolates, the number of roots ranged between a maximum of 33.3 for llyonectria sp2 (the single species that did not differ significantly from the control; 36.3) and a minimum of 20.7 for I. europaea, representing a 43% reduction in the number of roots. Among the isolates from other hosts, "C." pauciseptatum, I. macrodidyma and I. novozelandica did not differ statistically from the control (non-grapevine isolates from "C." pauciseptatum and I. novozelandica differed significantly from grapevine isolates), while inoculations with I. robusta resulted in the lowest number of roots (a 32% reduction).

The length of the longest root was significantly lower for all samples when compared to the control, ranging between a maximum of 33.8 cm for I. pseudodestructans (a 32% reduction from the control) and a minimum of 22.9 cm for I. estremocensis (54% reduction) for grapevine isolates, and between 38.2 cm for Ilyonectria sp1 and 24.9 cm for Ilyonectria sp2 for isolates from other hosts. Significant differences were recorded, however, for "C." pauciseptatum inoculations between grapevine (25.4 cm) and nongrapevine isolates (33.7 cm).

For grapevine isolates, the root dry weight of inoculated plants ranged between a maximum of 2.98 g for I. vitis (the single species that does not differ significantly from the control; 4.08 g) and a minimum of 1.28 g for I. lusitanica (a 69% reduction from the control). Among non-grapevine isolates, "C." pauciseptatum and Ilyonectria sp1 did not differ statistically from the control (and "C." pauciseptatum non-grapevine isolates differed significantly from grapevine isolates), while inoculations with Ilyonectria sp2 resulted in a root dry weight of 1.95 g (a 52% reduction).

Similarly, the number of shoot nodes ranged between a maximum of 13.2 for plants inoculated with I. vitis (the single species that did not differ significantly from the control; 15.9) and a minimum of 8.9 for plants inoculated with I. lusitanica (a 44% reduction from the control) for grapevine isolates, and between 12.5 for "C." pauciseptatum and 8.8 for Ilyonectria sp1 among isolates from other hosts. For each species, no significant differences were found between grapevine and non-grapevine isolates.

Shoot length was significantly shorter than that of the control for all samples, ranging between a maximum of 41.4 cm for llyonectria sp2 (a 21% reduction from the control) and a minimum of 24.3 for I. lusitanica (53% reduction) for grapevine isolates, and between 36.7 cm for I. torresensis and 21.8 cm for Ilyonectria sp1 for isolates from other hosts. Nongrapevine isolates had significantly higher values than grapevine isolates for several species, such as "C." pauciseptatum, I. liriodendri, I. macrodidyma and I. torresensis, while the opposite was recorded for Ilyonectria sp2.

The shoot dry weight ranged between a maximum of 0.96 g for llyonectria sp2 and a minimum of 0.41 g for I. lusitanica (a 57% reduction from the control) for grapevine isolates ("C." pauciseptatum, I. estremocensis, I. liriodendri, I. lusitanica and I. macrodidyma were significantly lower than the control) and of 0.45 g for Ilyonectria sp1 for non-grapevine isolates. Differences between grapevine and non-grapevine isolates were only recorded for Ilyonectria sp2 (0.96 g and 0.55 g, respectively).

Inoculated plants in the second experiment also revealed typical black foot symptoms, with significant reductions in root and shoot biomass as com pared to the control plants (Figure 2). Considering the species for which grapevine isolates were analysed, I. estremocensis was slightly more virulent than I. liriodendri and I. pseudodestructans, particularly in parameters concerning the aerial plant part, although the frequency of reisolation was significantly lower than that of I. liriodendri. Furthermore, results confirmed most non-grapevine isolates to be as virulent as grapevine isolates.

Discussion

Black foot disease symptoms recorded at the end of the experiments were associated with a reduction in plant growth and vigour, less shoot internodes and roots, and shorter and thinner shoots. These are illustrated by a reduction in the number of roots (up to 47%), shoot nodes (up to 49%), shoot length (up to 65%), length of the longest root (up to 66%), shoot dry weight (up to 80%), and root dry weight (up to 88%).

Frequency of reisolation was the least informative character, only separating the control plants and the isolates OL2 (llyonectria sp1) from Olea europaea and Cy230 (I. novozelandica) from Festuca duriuscula, from the remaining isolates. Traits related to the roots were slightly more informative than those related to the aerial plant parts, thus corroborating results from Alaniz et al. (2010).

In general, grapevine isolates from the species I. lusitanica, I. estremocensis, I. europaea and "C." pauciseptatum were the most virulent, while those from species such as I. novozelandica, I. pseudodestructans, I. vitis and llyonectria sp2 were the least virulent, with intermediate results for I. robusta, I. liriodendri, I. macrodidyma, I. torresensis and I. alcacerensis. For some species however, differences were recorded between characters related to the roots and to the aerial plant parts. Symptoms related to inoculations by I. lusitanica, I. estremocensis and "C." pauciseptatum isolates were equally prominent based on root and aerial part parameters. In contrast, symptoms caused by I. europaea, I. novozelandica and I. robusta isolates were more prominent on roots than on aerial parts, while symptoms of I. torresensis and I. macrodidyma were more noticeable on aerial plant parts. However, the effect of these pathogens in the aerial parts should be interpreted while taking into consideration that only ungrafted rootstocks were studied here. Experiments using grafted plants would be necessary to reach conclusions on the effect of these pathogens on the aerial parts of grapevine plants. In spite of this, the results obtained here reveal that different Ilyonectria species and "C." pauciseptatum induce diverse levels of severity on the aerial plant parts. This observation may be relevant in infected fields of rootstock mother-plants, because, most likely, the canes will be shorter, thinner and of poorer quality, thus compromising the later success of cuttings made from such vines.

A comparison among all isolates revealed isolates Cy243 (I. estremocensis), Cy197 (I. lusitanica), Cy23 (I. robusta), Cy238 ("C." pauciseptatum) and Cy128 (I. macrodidyma), all from grapevines, to be the most virulent, while the least virulent were isolates OL-CM3 ("C." pauciseptatum) from Olea europaea, Cy200 (llyonectria sp2) from grapevine, CBS 129081 (I. pseudodestructans) from grapevine, CBS 112593 (I. novozelandica) and Cy164 (I. liriodendri) from Malus domestica. Virulence to the roots varied among isolates, which in turn exhibit different effects on the aerial parts. Isolates Cy23 (I. robusta), Cy128 (I. macrodidyma), Cy152 (I. estremocensis), Cy196 ("C." pauciseptatum), CBS 110.81 (I. liriodendri, from Liriodendron tulipifera) or CBS 117824 (I. pseudodestructans, from Quercus sp.) showed high virulence in roots, but limited effects on the aerial parts. On the contrary, isolates CBS 129086 (I. torresensis), Cy250 (I. macrodidyma), CBS 537.92 (I. europaea, from Aesculus hippocastanum), CBS 159.34, and particularly isolate OL2 (llyonectria sp1, from Olea europaea) had low reisolation frequency and caused little effect on roots, but a very prominent effect on the above ground parts of inoculated plants. When isolate OL2 was inoculated on olive plants, it was found to be highly virulent (Cabral et al., unpubl. data), inducing not only aerial symptoms but also root and crown necroses. This indicates that llyonectria sp1 may be more host-specialized than the other species studied here, suggesting that although there are taxa with wide host ranges, host specialisation also occurs in some species of llyonectria. However, the unexpected pattern of symptoms produced by OL2 or other isolates, suggests that further work is required to fully elucidate the grapevine-Ilyonectria pathosystem. To date little information exists on the mechanisms of host infection and root colonization, as well as the concomitant mechanisms of host-defense response. In apple trees for example, it was hypothesized that the most virulent "Cylindrocarpon" isolates do not proliferate extensively within the host tissue, but rather cause damage to the host by the secretion of cell wall degrading enzymes or toxins (Tewoldemedhin et al., 2011).

For each fungal species, comparisons between grapevine and non-grapevine isolates could not suggest specific trends, with the notable exception of isolates from Olea europaea (and to some extent from Festuca duriuscula), which were always less virulent than grapevine isolates from the same species ("C." pauciseptatum, I. macrodidyma and I. torresensis). However, frequency of reisolation did not differ significantly to that of other isolates, suggesting that these isolates are fully capable of infecting and colonizing the inoculated plants. The capacity of isolates from hosts such as Actinidia chinensis, Fragaria x ananassa, Malus domestica and Quercus spp. to be as virulent as the grapevine isolates, including isolates from some of the most virulent species, such as I. europaea, raises the importance of the cross-infection potential of isolates from other hosts to grapevine, particularly for plants that are likely to precede grapevine in cultivation, either in a vineyard or nursery. In fact, a recent study addressing apple replant disease (Tewoldemedhin et al., 2011) revealed the involvement of species also pathogenic to grapevine in the present study, such as "C." pauciseptatum, I. macrodidyma and I. liriodendri, supporting their polyphagous nature.

Furthermore, many isolates of the I. macrodidyma species complex were obtained from roots of several monocotyledons and dicotyledons weed families sampled in Spanish vineyards. When inoculated on grapevines, these isolates were able to induce typical black foot disease symptoms (Agustí-Brisach et al., 2011). In addition to the hosts referred to above, therefore, weeds may represent an important inoculum source of I. macrodidyma s. lat. in vineyards.

Besides the importance of cross-infection potential as well as indications of host specificity, the present study also revealed that grapevine isolates from newly described species such as I. lusitanica, I. estremocensis and I. europaea are more virulent to grapevine than the species previously accepted to represent the main causal agents of black foot, such as I. liriodendri and I. macrodidyma.

Acknowledgments

We thank M.Sc. Teresa Nascimento, M.Sc. Ana Teresa Vaz, Mrs. Amélia Marques and Mrs. Olga Natividade for technical assistance. This article is part of a Ph.D. dissertation (Instituto Superior de Agronomia, Technical University of Lisbon, Portugal). This work was partially supported by Fundação para a Ciência e a Tecnologia, Portugal (grant number SFRH/BD/24790/2005; project PTDC/AGRAAM/ 099324/2008).

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Accepted for publication: January 20, 2012