The aim of this study was to investigate the occurrence of a new symptom called pepper spot on avocado and mango fruits caused by Colletotrichum gloeosporioides. A total of 400 C. gloeosporioides isolates were collected for molecular diversity and pathogenicity studies. Of these, 250 were from cv. Hass avocado fruit and 1 50 were from cv. Kensington Pride mango fruit. Of the isolates from avocado fruit, half were from anthracnose lesions on mature, ripening fruit and half were from pepper spot lesions on mature, green fruit. Avocado isolates were collected from five different orchards in eastern Australia (Bangalow, Cudgen, Duranbah, Green Pigeon, Mt Tamborine). Of the mango isolates, half were from anthracnose lesions on mature, ripening fruit and half were from the tear stain (similar to pepper spot) symptom on mature green fruit. Mango isolates were collected from three orchards in eastern Australia, two of which corresponded with orchards from which avocado isolates were collected (Bangalow and Green Pigeon) and one which was located a significant distance (Ayr, North Queensland) from all other farms.
Firstly, it was confirmed by Koch's postulates that C. gloeosporioides causes pepper spot on avocado fruit. Although mango fruit were not inoculated with C. gloeosporioides, the fungus was consistently isolated from excised tear stain spots.
Through DNA amplification fingerprinting of the C. gloeosporioides isolates, it was determined that fungal populations from avocado were highly heterogeneous. Culture studies also found that, under set laboratory conditions, some avocado isolates from both pepper spot and anthracnose symptoms were able to produce the sexual stage (teleomorph) of the fungus, Glomerella cingulata. Fungal populations from mango, on the other hand, were comparatively homogeneous and, under the same conditions, did not produce the teleomorph in culture. It was determined that, although all but one of the avocado isolates capable of producing the teleomorph clustered into a main avocado group (which also contained anamorphic isolates), there was no definitive relationship between the ability of the isolates to produce the teleomorph and their clustering patterns or pathogenicity.
This study supported reports that mango isolates are not found on other crops and usually are virulent only on mango. Isolates from avocado were genetically distinct from mango isolates and there were no genetically identical strains found on both fruit types, concluding that there is limited threat of avocado isolates infecting mango, and vice versa. Although this study did not include mango inoculations, it was shown that mango isolates were only weakly pathogenic on avocado and were in a distinct genetic population. It was suggested that the mango fungal population has most likely coevolved with the fruit, and may initially have had a geographic distribution limited to the centres of diversity of mango.
On the basis of DNA amplification fingerprinting studies, it was concluded that particular symptoms of each fruit type (avocado or mango) were not associated with different pathogen genotypes. The preharvest avocado pepper spot symptom was not caused by distinct strains of C. gloeosporioides compared to those causing postharvest avocado anthracnose. Similarly, the preharvest mango tear stain symptom was not caused by distinct strains of C. gloeosporioides compared to those causing postharvest mango anthracnose.
Using DNA fingerprints of isolates collected from a range of sites, it was possible to analyse patterns of isolates within and between orchards and crops. Cluster analysis grouped the majority of isolates according to geographic origin. However, even though the mango isolates from Ayr in northern Queensland were geographically isolated, there was no obvious population clade distinct from the remaining mango isolates from northern NSW. There were genetically identical populations of isolates within mango trees and mango orchards.
Investigations were carried out to see whether there was a relationship between DNA polymorphism and variation in pathogenicity (ability to cause disease on a given host) and aggressiveness (relative capacity to cause disease on a given host genotype) in the pathogen populations. Eighty C. gloeosporioides isolates were selected on the basis of geographic location, host, symptom type and specific location within an orchard (e.g. collected from adjacent avocado and mango trees). These isolates were screened for host specificity and pathogenicity as well as comparative aggressiveness by inoculating onto detached mature 'Hass' fruit and detached seedless 'Fuerte' fruit (referred to as "cocktail" avocados), leaf petioles of young grafted nursery trees, as well as 'Hass' avocado fruit and pedicels still attached to the tree.
On detached, ripening avocado fruit in the laboratory, there were no significant differences between the capacity of avocado anthracnose and avocado pepper spot isolates to cause anthracnose, nor were there significant differences between the capacity of mango anthracnose and mango pepper spot isolates to cause anthracnose on avocado. There was, however, a clear distinction in disease causing ability on detached fruit between mango isolates and avocado isolates, with disease incidence being lower after inoculation with mango isolates even under these artificial conditions. Some of the mango isolates were not pathogenic at all and many produced only a slight blackened stain on the fruit surface without further necrosis into the tissue, even after 1 0 days. Of the mango isolates causing anthracnose, there were variations in aggressiveness for individual isolates. Likewise, of the avocado isolates, levels of aggressiveness varied for individual isolates, although the outstandingly aggressive isolates based on mean lesion diameter were from pepper spot symptoms. When isolates were grouped according to the farm of origin, there were some significant differences in aggressiveness but isolates could not be distinguished by this characteristic. The main outcome of this experiment was that pepper spot isolates were as capable as anthracnose isolates of causing anthracnose in detached, ripening fruit.
Pepper spots were formed on unripe avocado fruit and pedicels on the tree at all stages of maturity. Likewise, pepper spots developed on petioles of nursery avocado trees, but not their leaves. Most isolates were pathogenic at the high inoculum levels (5x 1 06 conidia/mL) used in the experiments but they varied in their relative aggressiveness. When all isolates were grouped according to symptom of origin, significant differences were demonstrated between avocado anthracnose and avocado pepper spot isolates on petioles in the glasshouse and on unripe fruit in the field. Mango isolates were only weakly pathogenic on avocado and some were not pathogenic at all, and there were no significant differences in aggressiveness on avocado between mango anthracnose and mango pepper spot (tear stain) isolates.
When isolates were grouped according to the farm of origin, there were some significant differences. Avocado isolates (both pepper spot and anthracnose) from Green Pigeon showed low pathogenicity equivalent to the mango isolates in the glasshouse, but when inoculated in the field, were comparable to the rest of the avocado isolates. There were more and less pathogenic strains present in the pathogen populations from both mango fruit and avocado fruit but neither were restricted to anthracnose or pepper spot groupings. Generally though, of the most aggressive isolates, a higher percentage was from avocado pepper spot. When pathogenicity of individual isolates was compared with genetic clustering on the dendrograms, there were no correlations i.e. highly aggressive isolates did not group together. Factors affecting infection by C. gloeosporioides and the development of pepper spot in the field were explored. Rootstocks, nitrogen fertilisation levels, inoculum concentrations, fruit maturity, season and fruit pH were some of the influences studied. It was found that many factors influencing disease on avocado trees tended to have the same impact for both pepper spot and anthracnose disease incidence. Avocado fruit were inoculated with pepper spot isolates of C. gloeosporioides throughout the growing season from soon after fruit set until full fruit maturity to assess disease incidence during changing annual seasons (spring through to winter). Growing season and fruit maturity had some effect on pepper spot infection with the incidence of pepper spot infection increasing during the summer months. Disease incidence and severity also increased when fruit were inoculated with an increasing fungal spore concentration. This concurred with previous studies of avocado fruit inoculations by Coates (1991) and Alahakoon et al. (1994a). ........................