Fungal Pathogens Associated with Almond Decline Syndrome, an Emerging Disease Complex in Intensive Almond Crops in Southern Spain.

In 2016, an almond (Prunus dulcis) decline syndrome (ADS) emerged in intensive almond plantations in the Andalusia region (southern Spain), showing branch dieback, gummosis, and general tree decline. The aim of this work was to elucidate the etiology of this disease complex. For this purpose, surveys were conducted across the Andalusia region, and a wide collection of fungi was recovered from wood samples showing gum and internal discoloration. Representative isolates were selected and identified by sequencing ITS, TEF1, TUB, ACT, LSU, and/or RPB2 genes. The following fungal species were identified to be associated with the disease: Botryosphaeria dothidea, Diplodia corticola, Di. seriata, Dothiorella iberica, Lasiodiplodia viticola, Macrophomina phaseolina, Neofusicoccum mediterraneum, N. parvum, N. vitifusiforme, Diaporthe neotheicola, Dia. rhusicola, Dia. ambigua, Eutypa lata, E. tetragona, Eutypella citricola, Eu. microtheca, Fusarium oxysporum s.l., Pleurostoma richardsiae, Phaeoacremonium iranianum, Pm. krajdenii, Pm. parasiticum, and Cytospora sp. All isolates were tested for pathogenicity by inoculating detached or attached almond shoots. Di. corticola and N. parvum were the most aggressive species, showing the largest lesions and most gummosis in attached shoots. The results suggest that the species belonging to Botryosphaeriaceae play a key role in disease development, while the remaining identified species may act as secondary pathogens or endophytes. However, further research to determine the interaction between all these fungal species and other biotic and abiotic factors in the ADS progress is needed.

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide.

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.

Effects of Cultivar Susceptibility, Fruit Maturity, Leaf Age, Fungal Isolate, and Temperature on Infection of Almond by Colletotrichum spp.

Almond anthracnose, caused by Colletotrichum spp., is a reemerging disease in Spain. To date, little research has been conducted on the factors affecting this disease development. In this study, the effects of cultivar, fruit wounding and maturity, leaf age, fungal isolate, and temperature on almond infection by Colletrotrichum spp. were evaluated under laboratory-controlled conditions. Inoculations were performed using conidial suspensions of Colletrotrichum acutatum or C. godetiae. Disease severity was higher in wounded than in unwounded fruit. Based on observations of inoculated fruit, Ferraduel and Nonpareil were the most tolerant cultivars, while Tarraco and Penta were the most susceptible cultivars. Four categories of susceptibility (highly susceptible, susceptible, moderately susceptible, and resistant) were distinguished by using the cluster analysis statistical approach. Differences in susceptibility between young and old leaves were observed, but Nonpareil was consistently the most tolerant cultivar. Significant differences in virulence between C. acutatum and C. godetiae were observed in inoculated fruit, with C. acutatum being the most virulent. Disease development was more severe when inoculations were performed at the fruitlet stage or when the fruit were incubated at approximately 25°C, with respect to other maturity stages and temperatures evaluated. Natural fruit infections were also assessed. Cultivar susceptibility data were compared between laboratory tests and field observations. A significant positive linear correlation was obtained between the susceptibility of the common cultivars evaluated under the two conditions.

Variability in Susceptibility to Anthracnose in the World Collection of Olive Cultivars of Cordoba (Spain).

Anthracnose of olive (Olea europaea ssp. europaea L.), caused by Colletotrichum species, is a serious disease causing fruit rot and branch dieback, whose epidemics are highly dependent on cultivar susceptibility and environmental conditions. Over a period of 10 years, there have been three severe epidemics in Andalusia (southern Spain) that allowed us to complete the assessment of the World Olive Germplasm Bank of Córdoba, one of the most important cultivar collections worldwide.A total of 308 cultivars from 21 countries were evaluated, mainly Spain (174 cvs.), Syria (29 cvs.), Italy (20 cvs.), Turkey (15 cvs.), and Greece (16 cvs.). Disease assessments were performed using a 0-10 rating scale, specifically developed to estimate the incidence of symptomatic fruit in the tree canopy. Also, the susceptibility of five reference cultivars was confirmed by artificial inoculation. Because of the direct relationship between the maturity of the fruit and their susceptibility to the pathogen, evaluations were performed at the end of fruit ripening, which forced coupling assessments according to the maturity state of the trees. By applying the cluster analysis to the 308 cultivars, these were classified as follows: 66 cvs. highly susceptible (21.4%), 83 cvs. susceptible (26.9%), 66 cvs. moderately susceptible (21.4%), 61 cvs. resistant (19.8%), and 32 cvs. highly resistant (10.4%). Representative cultivars of these five categories are "Ocal," "Lechín de Sevilla," "Arbequina," "Picual," and "Frantoio," respectively. With some exceptions, such as cvs. Arbosana, Empeltre and Picual, most of the Spanish cultivars, such as "Arbequina," "Cornicabra," "Hojiblanca," "Manzanilla de Sevilla," "Morisca," "Picudo," "Farga," and "Verdial de Huévar" are included in the categories of moderately susceptible, susceptible or highly susceptible. The phenotypic evaluation of anthracnose reaction is a limiting factor for the selection of olive cultivars by farmers, technicians, and breeders.

Morphological, Pathogenic, and Molecular Characterization of Colletotrichum acutatum Isolates Causing Almond Anthracnose in Spain.

Almond anthracnose is a serious and emerging disease in several countries. All isolates causing almond anthracnose have been assigned to the Colletotrichum acutatum species complex, of which only C. fioriniae and C. godetiae have been associated with the disease to date. Here, we characterized Colletotrichum isolates from almond fruit affected by anthracnose in the Andalusia region. Two Colletotrichum isolates causing olive anthracnose were included for comparison. Morphological characteristics were useful for separating the isolates into groups based on colony morphology. Pathogenicity tests in almond, olive, and apple fruit showed differences in virulence and some degree of pathogenic specialization among isolates. Molecular characterization allowed clear identification of the Colletotrichum isolates tested. The olive isolates were identified as C. godetiae and C. nymphaeae, both previously identified in Andalusian olive orchards. Two phylogenetic species were identified among the almond isolates: C. godetiae, with gray colonies, which is well known in other countries, and C. acutatum, with pink-orange colonies. This species identification differs from those of pink-colony subpopulations described in other countries, which are C. fioriniae. Therefore, this study is also the first report of a new species of Colletotrichum causing almond anthracnose within the C. acutatum species complex.

Factors Affecting Infection and Disease Development on Olive Leaves Inoculated with Fusicladium oleagineum.

Infection and development of olive scab disease, caused by Fusicladium oleagineum, were evaluated on detached leaves and potted plants of the susceptible cultivar Picual in growth chambers and a shadehouse. An inoculum dose of 1 × 105 conidia per ml was selected from a range of densities tested, and it was used for all experiments. Infection occurred from 5 to 25°C, and disease severity was the greatest at ~20°C for wetness durations of 12 to 24 h and at ~15°C for longer durations. Based on a generalized form of the Analytis Beta model, the optimum temperature and minimum wetness duration for infection were 15.5°C and 11.9 h. Dry periods ≤78 h immediately after inoculation did not reduce disease incidence but did reduce disease severity. Disease severity was negatively correlated with leaf age. Disease incubation period was positively correlated with leaf age, with a minimum incubation period of 28 days in the youngest leaves. Inoculated plants that were incubated in a growth chamber or in a shadehouse had the same level of infection, but disease severity was lower in plants incubated in the growth chamber because many infections remained latent for 6 months after inoculation. The data in this study will be useful for the development of a forecasting system for olive scab epidemics.