Genetic variability within pathogenicity groups

There was no obvious relationship between genotype and pathogenicity in canes (Figure 3.9). Although the three isolates from Pathotype A with the highest ratio values (>2.0; Table 3.5) clustered in Clade III and seven out of ten isolates belonging to Pathotype C clustered in Clade IV, these isolates of the same pathotype all originated from the same nursery,

therefore, the clustering may also be due to origin. The remaining isolates from pathotype Groups A, B and C were randomly distributed among the four clades of the neighbour joining tree.

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Figure 3.9 Neighbour joining dendogram depicting patterns of genetic variability among 29 N. luteum isolates from different nurseries based on UP-PCR analysis using five primer combinations. The red dotted line indicates the changes at which the branches were defined into four major clades (roman numerals). Where letters on the right of the isolate numbers match, this indicates that the isolates were from the same plant sample. Isolates shaded in orange indicate clustering by pathotype and blue indicate clustering by nursery origin.

L70 A Nursery 3 L560A C Nursery 5 L523 B Nursery 5 L472 B Nursery 5 L86B A Nursery 3 L106C A Nursery 3 L89 B Nursery 3 L481 C Nursery 5 L110 B Nursery 3 L113 B Nursery 3 L493 B Nursery 5 L248 A Nursery 7 L114 A Nursery 3 L460 B Nursery 5 L85B B Nursery 3 L105C B Nursery 3 L228D A Nursery 7 L229D A Nursery 7 L244 A Nursery 7 L456 A Nursery 5 L465 C Nursery 5 L559A C Nursery 5 L483E B Nursery 5 L555 C Nursery 5 L501 C Nursery 5 L552 C Nursery 5 L485E C Nursery 5 L468 A Nursery 5 L557 C Nursery 5

Pathotypes Nursery source

I

II

III

IV

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3.2.4 DISCUSSION

This study used UP-PCR analysis to investigate the genetic variability among isolates of

N. luteum collected from different nurseries that showed different degrees of pathogenicity on green shoots and canes. The analysis produced high numbers of polymorphic bands that allowed the differentiation of different N. luteum isolates and showed the genetic diversity of the nursery isolates of this species.

The analysis of multiple isolates from five plant samples showed the potential for pathogen movement, since the isolates recovered from above (L228) and below (L229) the graft union in one healthy grafted plant were of one genotype. A study by Gambetta et al. (2009) using xylem-mobile dyes and the xylem-limited bacterial pathogen Xyllela fastidiosa demonstrated that xylem conduits bridge the graft unions, so they could allow the passive movement of pathogens. Therefore, these open conduits may facilitate the movement of the N. luteum

infections from either rootstock or scion and allow for development of systemic infections in young vines. However, it is also possible that multiple infections by the same genotype can occur in the same plant by splash dispersal from a single source of conidia from a nearby plant surface.

The analysis also demonstrated intra-plant genotype variation, indicating separate infection events of an individual plant sample. One grafted plant and three cuttings contained multiple isolates of different genotypes, indicating that they were infected by inoculum from different sources within the mothervine blocks or the propagation systems. Intra-vine variability was also observed among Ph. chlamydospora isolates suggesting that multiple introductions had occurred (Pottinger et al., 2002; Mostert et al., 2006). Given that multiple infections by genetically distinct isolates are occurring, investigations were done (Chapter 5) into the sources of botryosphaeriaceous species inoculum and their potential dispersal mechanisms within the nursery system as this knowledge could enhance the development of control strategies.

Inter- and intra-nursery comparisons showed that different genotypes were present within a nursery and in different nurseries in New Zealand. Except for Nursery 7, no clonal isolates were found among those obtained from Nursery 3 and Nursery 5. Furthermore, the

genetically distinct isolates from different nurseries were randomly distributed among three clades suggesting they are highly diverse and that a mechanism exists for either frequent incursion of new isolates to the nursery and/or the presence of an active recombination mechanism. However, nine of the isolates from Nursery 5 were clustered in Clade IV indicating that these isolates were closely related. Baskarathevan et al. (2009) found a high degree of genetic variability among N. parvum isolates from New Zealand vineyards with

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higher inter-vineyard variability than intra-vineyard. Che-Omar (2009) also found similar results with N. parvum isolates from six blueberry farms in New Zealand; the 30 isolates from three individual plant samples from each farm were each of unique genotype. These results are in contrast with genetic variability studies of other fungal pathogens that have

predominantly asexual reproductive mechanisms. For example, Pottinger et al. (2002) detected low genetic variation among New Zealand populations of Ph. chlamydospora, for which they found only nine genotypes out of the 45 isolates analysed using UP-PCR. They also found that these NZ isolates shared high genetic similarity with the Italian isolates analysed. A study by Mostert et al. (2006) also showed low genetic variation among

Ph. chlamydospora isolates from South Africa, which comprised 19 genotypes among 68 isolates analysed using amplified fragment lengths polymorphism (AFLP). Obanor et al.

(2010) also observed low levels (13%) of genetic variation among S. oleagina populations in New Zealand and showed great similarity with isolates from Australia and Italy using UP- PCR analysis. A study by Alaniz et al. (2009) also showed low genetic variability among

C. liriodendri and C. macrodidymum isolates.

Botryosphaeriaceous species were concluded to be predominantly asexual in their

reproduction since their teleomorph stages were rarely encountered in the field (Jacobs & Rehner, 1998; Phillips, 2002; Slippers & Wingfield, 2007). Previous research on D. sapinea

also supported this hypothesis since spore-trapping studies had shown that it spread exclusively via conidia (Swart et al., 1991) and genetic diversity studies using simple

sequence repeat (SSR) markers had provided some evidence that this species is obligately asexual (Burgess et al., 2004). The high numbers of clonal isolates observed among species of B. dothidea (Michailides, 1991; Ma et al., 2004), L. theobromae (Burgess et al., 2005) and

N. parvum (Slippers & Wingfield, 2007) were consistent with species that are predominantly asexual in their reproduction. The high genetic variability of N. luteum isolates found in this study and in the studies reported by Baskarathevan et al. (2011) and Che Omar (2009) were, therefore, unexpected. These suggest that despite the widespread asexual reproduction observed among botryosphaeriaceous species in New Zealand, genetic recombination is occurring in nature, resulting is highly diverse populations. According to Carlile et al. (2001), although 20% of all known fungi reproduce purely asexually, some genetic recombination has been observed in most of these species. Unlike other organisms, fungi have novel mechanisms of genetic exchange and recombination that are not limited to sexual

reproduction, which include heterokaryosis and parasexualism (Anderson & Kohn, 1998). However, parasexual crosses occur less frequently than sexual ones and their occurrence in natural populations has not been proven conclusively (Anderson & Kohn, 1998). According to Agrios (2005), while the frequency and degree of variability between asexually produced individuals are lower, the number of individuals produced asexually is so high that the total

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amount of variability produced by relatively few individuals in the asexual populations is probably greater than the variability from those sexually produced populations. Since species with abundant genetic variability are more likely to survive the challenges of a changing environment (Carlile et al., 2001), the high genetic diversity among N. luteum isolates indicates that this species is likely to be more resilient in changing conditions and would be more difficult to control than those pathogens that are highly clonal and have low genetic variability. Therefore, this genetic diversity should be taken into consideration in developing control strategies for this pathogen.

The high genetic diversity of N. luteum populations within nurseries may be partially due to the use of propagation materials from different sources. Although most nurseries grow their own rootstock material, their scion materials are usually provided by different vineyards through pruning (Verstappen, pers. comm., 2009). However, some nurseries also produce the majority of scion wood propagation materials in their own mothervine blocks. The movement of plant materials from vineyards to nurseries or one nursery to another may introduce new pathogen populations, thereby increasing the genetic diversity of the pathogen within the nursery. However with Ph. chlamydospora, which is known to spread through infected plant materials from nurseries (Fourie & Halleen, 2004b; Whiteman et al., 2007; Aroca et al., 2010), high clonality and low genetic variation has been reported. This may be associated with vegetative reproduction, since infection of cuttings is sometimes due to endophytic colonisation directly from the mothervines (Edwards & Pascoe, 2004; Whiteman

et al., 2007), but may also be due to the fact that this species reproduces in the field predominantly through asexual processes (Pottinger et al., 2002; Mostert et al., 2006). Pottinger et al. (2002) further found that Ph. chlamydospora isolates from different genetic groups belonged to one mycelial compatibility group, further confirming that all isolates were genetically similar. Baskarathevan (2011) found four vegetative compatibility groups among

N. parvum isolates from New Zealand vineyards, indicating the genetic variability of this species. Therefore, it is possible that botryosphaeriaceous species employ a different mechanism in genetic exchange and recombination than Ph. chlamydospora. This hypothesis can be confirmed by doing vegetative compatibility tests among N. luteum

isolates to determine if hyphal anastomosis occurs among isolates and establish the mechanisms for genetic recombination.

This study found no correlation between genotypes and pathogenicity in canes since the highly, moderately and weakly pathogenic isolates were randomly distributed in four clades. To further confirm this result, the genetic variability results were further compared with the pathogenicity of isolates on green shoots, but similar trends were observed. The isolates that were highly pathogenic and weakly pathogenic on green shoots were randomly scattered

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throughout the neighbour joining tree indicating that pathogenicity on green shoots was also not correlated with genotype. Although some other genetic variability studies found some correlation between genotype and virulence, it is important to note that this study was developed in the reverse order to the studies of Baskarathevan (2011) and Alaniz et al.

(2009). Those studies investigated the genetic variability of a wide range of isolates and then selected groups of isolates for pathogenicity tests on detached green shoots from the

branches of the neighbour joining tree they had generated and Baskarathevan (2011) further confirmed the results using rooted canes. Their pathogenicity studies also inoculated and assessed the test plants in one batch using similar tissues.

While the UP-PCR method used in this study was effective in showing the genetic diversity of different N. luteum isolates, this method was not able to provide any conclusive correlations between pathogenicity and genotypic variability. The lack of correlations observed may be due to the limitations of UP-PCR primers which according to Bulat et al. (1998), are primarily designed to target intergenic and non-coding areas of the genome. Since pathogen virulence is generally regulated by a single or multiple genes (Agrios, 2005), these specific areas of the genome were most likely not targeted by the UP-PCR primers. Therefore, the RAPD and AFLP methods that randomly target different regions of the genome (Hu et al., 1995; McDonald, 1997) may be more appropriate tools in investigating relationships between pathogenicity and genotypes.

In summary, high levels of genetic variability were observed among isolates of N. luteum

from different nurseries with only two clonal isolates found which was unexpected since previous reports have indicated that this group of fungi primarily reproduce asexually in the field and that other studies have shown some botryosphaeriaceous species to have many clonal isolates. The high genetic variability indicates that N. luteum can employ different mechanisms of genetic exchange. In addition, the presence of different genotypes within plants and nurseries, and between nurseries, indicated that infections may occur from different sources of inoculum. The high genetic diversity of isolates in this fungal group indicates that they might have a greater capacity to survive in changing environmental conditions, potentially making them more difficult to control, compared to those pathogens that are less diverse.

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