Bernardo A. Latorre

The effect of temperature and wetness duration on infection and a warning system for European canker (Nectria galligena) of apple in Chile

European canker (Nectria galligena Bres.) is a major disease of apple in Chile, mainly producing cankers on twigs and branches. Disease incidence over 50% has been reported on ‘Richard Red’ apples in Southern Chile. In vitro, conidial germination of N. galligena was low at 61C and 321C, and optimum germination temperature was estimated between 201C and 251C. Ascospore germination was low at 51C and rapidly increased as temperature increased from 51 Ct o 201C. Germination rate, calculated as germination frequency over time at 201C, was 2.3 times faster for ascospores than conidia, and germination of ascospores increased 2.6 times faster than conidia as temperature increased from 51 Ct o 201C. Infection caused by conidia of N. galligena through leaf scars was significantly dependent on temperature and free moisture duration. However, regardless of the wetness duration no infection was obtained at 51C. This is in agreement with the lack of conidial germination obtained below 61C by in vitro studies. Disease incidence increased linearly with temperatures between 5 and 201C. Based on our results, a 2-h moisture period was enough to promote a significant infection at 201C but longer moisture periods were needed at lower temperatures. An infection warning system, based on the analysis of weather parameters, was developed and implemented in the predictive software PatFrut r . Over two seasons, the warning system was an effective tool for determination of the need of fungicide treatments against European canker. Significant differences in disease incidence and severity were obtained reducing disease incidence from over 24% in 1999 to 4.6% when treatments were schedule according to the model program. Differences in severity, but not disease incidence were found between the model program and the standard program. This forecast model assumed that conidia, produced during leaf fall were not a limiting factor. It also assumed that susceptible leaf scars were continuously available between March and July. The protective fungicide treatments applied during leaf fall significantly reduced infection in the next season suggesting that leaf scars are important infection courts for N. galligena on apples. r 2002 Elsevier Science Ltd. All rights reserved.

Ochratoxigenic Aspergillus species on grapes from Chilean vineyards and Aspergillus threshold levels on grapes.

This study reports the incidence of ochratoxigenic strains of Aspergillus on Chilean grapes (Vitis vinifera) and wineries, and production of OTA levels in wines with grapes having different levels of contamination with OTA-producing Aspergillus carbonarius was studied. A. carbonarius, A. niger, A. niveus, A. paradoxus, A. versicolor, A. wentii, and A. westerdijkiae were identified on apparently healthy clusters of red and white grape cultivars. However, A. carbonarius and A. niger were the most frequently identified species, more abundant on red than white grape cultivars. Aspergillus spp. populations increased between veraison and harvest, but the isolation frequencies were relatively low over the entire growing season. At the winery, A. carbonarius, A. niger and A. westerdijkiae were occasionally found in the air, exclusively during winemaking. OTA-producing strains were only found among isolates of A. carbonarius, A. niger, A. wenti, and A. westerdijkiae, producing 2 to 17 microg/L of OTA in liquid medium; however, A. westerdijkiae produced the highest OTA concentration in vitro. Red wines elaborated with 0.5% of grapes infected with an OTA-producing strain of A. carbonarius (Aspuc-SB36) exceeded the 2 microg/L of OTA tolerance established for wines by the European Community. Therefore, a threshold below 0.5% infected berries is proposed for red wines. ELISA tests proved to be useful for detecting OTA in broth culture as in wine samples.

Pre- and post-infection activity of new fungicides against Botrytis cinérea and other fungi causing decay of table grapes

R.A. Serey, R. Torres, and B.A. Latorre. 2007. Pre- and post-infection activity of new fungicides against Botrytis cinerea and other fungi causing decay of table grapes. Cien. Inv. Agr. 34(3):215-224. Pre- and post-harvest diseases restrict table grape production and exports (Vitis vinifera L.) in Chile, with the most important disease being grey mold (Botrytis cinerea). In addition, rot due to Aspergillus niger, Cladosporium herbarum, Penicillium expansum, and Rhizopus stolonifer frequently occurs. The pre- and post-infection activity of fungicides against r these pathogens was studied on Thompson Seedless table grapes. Detached, mature, berries were used, and inoculations were performed with 20 μL of a 10 6 spores·mL -1 suspension placed on three punctures aseptically made at the calyx end of each berry. Fungicides used (per liter) were boscalid (600 mg), boscalid (200 mg) + pyraclostrobin (100 mg), boscalid (200 mg) + kresoxim methyl (100 mg), cyprodinil (60 mg) + fl udioxonil (40 mg), BAS 600 KBF (100 mg) + metrafenone (150 mg), BAS 600 KBF (200 mg) + boscalid (300 mg), BAS 600 KBF (100 mg) + pyraclostrobin (100 mg), and captan (400 mg). Each fungicide was applied either by drop (12 μL·berry -1 ) placed on three punctures made with a sterile hypodermic needle or by 60 s immersion. Berries were then incubated in humid chambers at 20oC. The pre-infection (protection) activity of the fungicides varied considerably among the pathogens tested and was found to be signifi cant (p < 0.001) and, with one exception (A. niger), it was signifi cantly (p < 0.002) affected by the application method. The interaction between fungicide and application method was only signifi cant (p < 0.001) for R. stolonifer at 48 h post treatment. In general, pre-infection activity gave 0 to 4 days protection after drop applications and 0 to 21 days after immersion treatments. The post-infection (curative) activity varied among pathogens and fungicide treatments. However, it was always below 24 h.

Characterization of Cladosporium Rot in Grapevines, a Problem of Growing Importance in Chile

Cladosporium rot (Cladosporium spp.) of grapevine (Vitis vinifera) is a common disease in Chile, particularly in Cabernet Sauvignon and other red wine grape cultivars. It is favored by delayed harvest to obtain the phenolic maturity necessary for high-quality red wine. This study expands on previous investigations of the specific causal agents, the histopathological host:pathogen relationship, and the population dynamics of Cladosporium spp. during the seasonal development of grape clusters. Over 100 isolates were obtained and identified as C. cladosporioides and C. herbarum, confirming previous results. The identity of a subset of isolates was confirmed by molecular analysis. Isolates of both C. cladosporioides and C. herbarum from grapevines were pathogenic on inoculated table grapes and wine grapes. These pathogens were reisolated, fulfilling Kochs postulates. Berry injuries and total soluble solids content above 15% were necessary for Cladosporium spp. to infect wine grapes. The mycelia of C. cladosporioides and C. herbarum grew between 0 and 30°C, but no growth was obtained at 35°C in vitro. The histological studies showed that Cladosporium spp. superficially colonize mature V. vinifera berries, invading the epidermis but scarcely penetrating the hypodermis. The Cladosporium populations obtained on apparently healthy berries of V. vinifera cvs. Cabernet Sauvignon and Chardonnay were significantly larger (P = 0.05) than the populations obtained under similar conditions on berries of V. champini cv. Ramsey and hybrids Kober 5BB and Couderc 1613. Considering the importance of Cladosporium rot in Chile compared with other grape production areas, the development of control strategies is needed to prevent high disease severity, which affects both yield and wine quality.

Characterization of Diaporthe australafricana and Diaporthe spp. Associated with Stem Canker of Blueberry in Chile

Stem canker and dieback are important factors that limit the longevity and reduce the yield of blueberry (Vaccinium spp.) in Chile. In this study, species of Diaporthe associated with blueberry were isolated and identified. The internal transcribed spacer (ITS) regions of ribosomal DNA of 30 isolates and the translation elongation factor 1-α (EF1-α) of 14 isolates were sequenced, analyzed, and compared with their morphological and pathological characteristics. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Diaporthe ambigua, D. australafricana, D. neotheicola, D. passiflorae, and Diaporthe sp. 1. However, morphology alone was insufficient to identify these species. The combined analysis of ITS and EF1-α gene sequences grouped the Chilean blueberry isolates in the same five groups obtained in the ITS analysis. Pathogenicity tests conducted with attached and detached blueberry shoots (<1 year old) and stems (1 to 2 years old) confirmed that isolates of these Diaporthe spp. were pathogenic. The symptoms were reproducible and consisted of necrotic reddish-brown cankers on blueberry shoots and stems. These isolates were capable of infecting blueberry fruit, causing a soft decay, suggesting that they were tissue nonspecific and were also pathogenic on shoots of apple, grapevine, and pear. D. australafricana was the most frequently isolated species and D. ambigua, D. australafricana, and D. passiflorae were highly virulent in shoots, stems, and fruit of blueberry. This study showed that at least four species of Diaporthe are primary pathogens, capable of causing stem canker symptoms on blueberry, and this is the first report of D. ambigua, D. neotheicola, and D. passiflorae attacking this host.

Riesgo de oídio (Erysiphe necator) de la vid en relación con el desarrollo de los racimos

En los ultimos anos, el oidio (Erysiphe necator) de la vid (Vitis vinifera) ha tenido especial importancia economica en la zona central y norte de Chile. Epidemias se desarrollan a partir del micelio latente que sobrevive en yemas infectadas la temporada precedente y/o a partir de cleistotecios desarrollados en hojas u otros organos infectados de la vid. La susceptibilidad de los racimos varia a traves de la temporada, siendo muy susceptibles las bayas jovenes y resistentes en la medida que maduran. El riesgo de infeccion se puede pronosticar en funcion de la temperatura maxima del aire. Con el proposito de mejorar la efi cacia del control del oidio, se realizo este trabajo con el objetivo de estudiar el riesgo de infeccion en funcion del desarrollo del racimo. Los resultados obtenidos demostraron que las aplicaciones fungicidas (kresoxim-metil, 65-70 mg?L-1 or miclobutanil, 24 mg?L-1) aplicadas a inicios de plena floracion (estadio 23) proporciono el mejor control del oidio en los racimos de vides cvs. ?Criolla?, ?Pedro Jimenez? y ?Semillon?. Por el contrario, la incidencia y severidad invariablemente aumento en racimos tratados en otros estadios. Esto se relaciono con la existencia de temperaturas del aire muy favorables al desarrollo de oidio, las que predominaron durante la floracion, determinando indices de riesgo de infeccion moderados a altos. En conclusion, los estadios del desarrollo comprendidos entre inicios de la fl oracion y bayas pequenas (estadios 19 a 29) fue el periodo mas critico para el desarrollo del oidio. Por lo tanto, tratamientos fungicidas aplicados durante la floracion son indispensables para controlar esta enfermedad.

Identification and Characterization of Botrytis Blossom Blight of Japanese Plums Caused by Botrytis cinerea and B. prunorum sp. nov. in Chile

Blossom blight is a destructive disease of plums (Prunus salicina) when humid and temperate weather conditions occur in Chile. Disease incidence ranging from 4 to 53% has been observed. Symptoms include light brown petal necrosis, starting as light brown mottles or V-shaped necrosis at the margins of the petals, progressing to the stamen and pistils. In this study, the etiology of blossom blight of plums was determined. High- and low-sporulating isolates of Botrytis were obtained consistently from blighted blossoms and apparently healthy flowers of plums. Based on colony morphology, conidial production and molecular phylogenetic analysis, these high- and low-sporulating isolates were identified as B. cinerea and B. prunorum sp. nov., respectively. Phylogenetic analysis of the genes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) grouped B. prunorum isolates in a single cluster, distantly from B. cinerea and other Botrytis species. The phylogenetic analysis of necrosis and ethylene-inducing protein (NEP1 and NEP2) genes corroborated these results. Analysis of the internal transcribed spacer region and large-subunit (26S) ribosomal DNA and detection of Boty and Flipper transposable elements, were not useful to differentiate between these Botrytis species. Both species were pathogenic on plum flowers and the fruit of plums, apples, and kiwifruits. However, B. prunorum was less virulent than B. cinerea. These pathogens were re-isolated from inoculated and diseased tissues; thus, Kochs postulates were fulfilled, confirming its role in blossom blight of plums. B. cinerea was predominant, suggesting that B. prunorum may play a secondary role in the epidemiology of blossom blight in plums in Chile. This study clearly demonstrated that the etiology of blossom blight of plums is caused by B. cinerea and B. prunorum, which constitute a species complex living in sympatry on plums and possibly on other stone fruit trees.

The effect of temperature on infection and a warning system for pear blossom blast caused by Pseudomonas syringae pv. syringae

Blossom blast caused by Pseudomonas syringae pv. syringae van Hall, is a major disease of pears (Pyrus spp.) in Chile. Freezing temperatures may predispose pears to infection; however, our results demonstrated that blossom blast infection might occur in the absence of frosts, at temperature higher than 5°C, provided that the appropriate inoculum concentration, moisture levels and the susceptible bloom stages were available. Regardless of the flower bud stage development, blossom blast was lowest at 5°C and highest at 20°C. The first symptoms appeared after 2–3 days at 20°C, and were delayed 2 days at 5°C. A significant and positive linear regression best explained the relationship between disease incidence and temperature. A warning system, based on temperature and free moisture conditions, was developed and found to be useful for prediction of blossom blast infection in the field. Antibiotic treatments applied after each warning significantly (p<0.05) reduced blossom blast on Packhams Triumph pears.

Severe Outbreaks of Bunch Rots Caused by Rhizopus stolonifer and Aspergillus niger on Table Grapes in Chile

Severe outbreaks of bunch rots (BR) have occurred recently during harvest of table grapes (Vitis vinifera L.) in Chile. Previously, BR was almost exclusively associated with Botrytis cinerea Pers.:Fr. (2,3); however, in 2000 to 2002, BR symptoms were associated with black molds and possibly nonfilamentous yeasts and bacteria. Cvs. Thompson Seedless, Flame Seedless, Ruby Seedless, and Red Globe were severely affected. Symptoms start at the pedicels as soft, watery rots that partially or completely decay infected berries. Longitudinal cracks are produced, a black mold usually develops along the crack fissures, and the skin of the berry turns light gray. Isolations on potato dextrose agar acidified with 1 N lactic acid (APDA) at 0.5 ml/liter, consistently yielded Rhizopus stolonifer (Ehrenb. ex Fr.) Vuillemin and Aspergillus niger Tiegh. R. stolonifer on APDA produced a white-to-gray aerial and nonseptate mycelium, black and globose sporangia with an elliptical collumela, one-celled, globose to oval, striated, almost hyaline sporangiospores, rhizoids, and stolons. A. niger produced septate mycelium. Single-celled, black, rough walled, globose conidia developed on short chains on the second phialides at the tip of globose, upright conidiophores. Mature (soluble solids >16%) detached berries of cv. Thompson Seedless were inoculated with sporangiospores (≈107 spores per ml) of R. stolonifer isolates RS6, RS52, RS73, and RS79 and conidia (≈108 conidia per ml) of A. niger isolates AN12, AN69, and AN75. When berries were aseptically punctured with a sterile hypodermic syringe prior to inoculation, 60 to 86.7% and 42.5 to 100% of berries were infected with R. stolonifer and A. niger, respectively, and both developed BR symptoms (significantly different from control berries) after 48 h in humid chambers at 23°C. Injuries were needed for infection since no infection or only 23.3% of noninjured berries were infected with R. stolonifer and A. niger, respectively. For both pathogens, there was a significant (P < 0.043) interaction between isolates and the presence or absence of injuries. Both pathogens were successfully reisolated on APDA. Fungicide sensitivity tests were performed on detached cv. Thompson Seedless berries challenged by placing an ≈6 μl-drop of inoculum suspension (106 or 107 spores per ml of R. stolonifer isolate RS52 and A. niger isolate AN12, respectively) on injured berries. Pyraclostrobin (0.067 mg/ml) mixed with nicobifen at 0.134 mg/ml (BAS 516 01 F at 0.201 mg a.i./ml, BASF) and copper oxide at 1.2 mg/ml (Cuprodul 60 WP, Quimetal Chile) significantly (P < 0.01) inhibited infection (100% control) by R. stolonifer and A. niger. R. stolonifer was completely controlled by dicloran at 1.88 mg/ml (Botran 75 WP) and partially controlled by captan at 1.6 mg/ml (Captan 80 WP), but A. niger was not controlled by either fungicide. To our knowledge this is the first report of R. stolonifer causing BR of table grape in Chile (4). The severe outbreaks may be associated with warm weather conditions during harvest and injuries caused by birds, insects, or cultural practices. Infection caused by R. stolonifer or A. niger may be followed by sour rot organisms (yeasts or bacteria), as has been suggested elsewhere (1,2). References: (1) E. Gravot et al. Phytoma 543:36, 2001. (2) W. B. Hewitt Page 26 in: Compendium of Grape Diseases, American Phytopathological Society, St. Paul, MN, 1994. (3) B. A. Latorre and G. Vásquez. Aconex (Chile) 52:16, 1996. (4) F. Mujica and C. Vergara. Flora Fungosa Chilena. Universidad de Chile, Facultad de Agronomiacute;a, Santiago, Chile, 1980.

Low Occurrence of Patulin-Producing Strains of Penicillium in Grapes and Patulin Degradation during Winemaking in Chile

Penicillium expansum has emerged as the cause of storage decay of table grapes (Vitis vinifera) and has been frequently isolated from apparently healthy clusters of grapes in Chile. The objectives of this study were to identify patulin-producing strains of Penicillium associated with winegrapes and wineries in Chile and to determine the potential presence of patulin in wines made with grapes infected with P. expansum. In this study, P. brevicompactum, P. expansum, and P. glabrum were identified on apparently healthy grape clusters and in the air of vineyards and wineries. Of 132 Penicillium isolates, 4 P. brevicompactum and 11 P. expansum strains were patulin-producing, determined by HPLC-UV/DAD. Patulin was also detected in Cabernet Sauvignon musts produced with grapes contaminated with a patulin-producing strain of P. expansum. However, patulin concentrations decreased during fermentation by 67.3 to 83.3%. Overall, the frequency of P. expansum isolation from grapes was relatively low; thus, considering the rapid degradation of patulin produced during fermentation, the risk of patulin contamination of bottled wine appears to be low.