Inoculum quantification of canker-causing pathogens in prune and walnut orchards using real-time PCR.

AIMS: A real-time quantitative PCR (qPCR) assay was established to quantify the inoculum densities in the air and rainwater for six canker-causing pathogen groups in prune and walnut orchards in California. METHODS AND RESULTS: The previously published DNA primers to target six pathogen groups including Botryosphaeria dothidea, Cytospora spp., Diplodia spp., Lasiodiplodia spp., Neofusicoccum spp. and Phomopsis spp. were used in a qPCR assay. Air samples from Burkard spore traps and rain samples from special rain collector devices were collected periodically from various prune and walnut orchards. Using the qPCR approach, we were able to quantify the concentrations of these pathogen groups in rainwater and air samples and study the dynamics of pathogen inoculum in orchards showing severe canker potential. Phomopsis spp. and Diplodia spp. were not found in all rain samples in prune orchards, although they were detected in the 2016 in the walnut orchard. The other four pathogen groups were quantified at varying concentrations in the prune and walnut orchards. Cytospora spp. in some cases showed higher concentrations in the rainwater in prune orchards. CONCLUSIONS: The rainy season during winter and early spring is a highly risky period of time for infection by the pathogens when the inoculum of these pathogens can easily spread by air and rain water, thus serving as an important inoculum source for disease initiation. The different studied pathogen groups showed different concentrations during the growing season, indicating the complexity of the components of canker-causing species in various tree crops. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the applicability of the qPCR assay in the quantification of inoculum in tree orchards to help reveal the mechanisms of canker disease epidemics and to help design disease management strategies.

Effects of Wound Size, Amount of Sap, and Number of Blighted Nuts on Infection of Pistachio Organs by Neofusicoccum mediterraneum.

Laboratory and field studies were conducted to determine the effects of wounding of nut exocarp, susceptibility period after wounding, and sap nut on infection of pistachio nut by Neofusicoccum mediterraneum, the main causal agent of panicle and shoot blight of pistachio. Under controlled conditions and in the field, detached nuts were inoculated with a conidial suspension 30 min before or after wounding. In addition, a 30-µl drop of pistachio sap was placed on the surface of noninjured nuts 30 min before or after they were wounded and then inoculated. Wounding increased the disease severity under both controlled and field conditions. The addition of sap increased the susceptibility of nuts under controlled conditions but not in the field, possibly due to dried sap blocking the pathogen infection. When nuts of Kerman, Kalehghouchi, and Golden Hills pistachio were wounded and inoculated at different time periods after wounding; the nuts of the three cultivars were highly susceptible to pathogen infection during at least the first 24 h after wounding. Under field conditions, there was not a clear effect of increasing the number of inoculated nuts per panicle or the inoculation position (basal or apical) in killing (blight) of the panicle. Conversely, inoculations conducted with mycelial plugs resulted in higher disease, increased the proportion of dead panicles, and resulted in faster symptom expression than inoculations conducted with a conidial suspension. To determine the temporal infection pattern, leaves and panicles were regularly collected from different orchards from 2004 to 2007 and the pathogen was isolated on medium. Important differences in latent infection were detected between years and orchards, with nut and rachis being, in general, the tissues most susceptible to infection. Results of this study help in better understanding the dynamic of infection and colonization of pistachio by N. mediterraneum.

Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops.

AIMS: To develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. METHODS AND RESULTS: This study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. CONCLUSIONS: For Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 5·60 to 6·94 during the 16 days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants.

First Report of Botryosphaeria rhodina Causing Shoot Blight of Pistachio in California.

In the summers of 2000 and 2001, shoot blight was observed in pistachios (Pistacia vera L.) grown in Kern County, California. Black, necrotic lesions developed at the base of shoots originating from contaminated or partially infected buds. Infection moved upward resulting in a progressive wilting and blighting of leaves. Leaf blades on infected shoots withered, and petioles became necrotic. Symptoms have been considered characteristic of infection by Botryosphaeria dothidea (Moug.:Fr.) Ces. & de Not., but this pathogen causes panicle and shoot blight of pistachio (1). However, there were no symptoms of any fruit panicle infections on trees we observed. Isolations on acidified potato dextrose agar from the base of blighted shoots in both years revealed a fast-growing fungus producing pycnidia which was identified as the anamorph Lasiodiplodia theobromae (Pat.) Griffon & Maubl. of B. rhodina Berk. & Curt. Arx. Identification of the pathogen was based on characteristic dark brown, oval pycnidiospores with striations on the surface of the spore along the long axis. Pathogenicity tests were performed on 12 Kerman pistachio trees grown at Kearney Agricultural Center, in Parlier, CA, using three isolates recovered from pistachios grown in two locations. Six to 16 current season shoots of pistachio trees (1 to 2 shoots per tree) were wounded with a 5-mm-diameter cork borer, and a mycelial plug of 5-day-old cultures of B. rhodina was inserted in each wound. Shoots were wrapped with Parafilm to prevent desiccation of inoculum. Six other shoots (one per tree) were inoculated similarly with mycelial agar plugs of a pistachio isolate of B. dothidea and served as positive controls, while six similar shoots were inoculated with only agar plugs and served as negative controls. Wilting of lower leaves in the majority of inoculated shoots started within 4 days for B. rhodina and 7 days for B. dothidea. Depending on the isolate of B. rhodina, 1 to 5 shoots and 50 to 80% of leaves were blighted within 7 days after inoculation. All inoculated shoots were left on the trees until 3 to 4 months after inoculation, pruned and assessed again. For inoculations done in September 2001, 33 to 71% of shoots were blighted, and the rest had cankers ranging from 22.5 to 28 mm long and 13.5 to 23.5 mm wide. A majority (67 to 100%) of shoots had pycnidia of the pathogen present. For inoculations done in October 2001, none of the shoots was blighted, but cankers ranged from 5 to 55.4 mm long and 6 to 22 mm wide and 33.3 to 100% developed pycnidia. B. rhodina was isolated from all inoculated shoots but not from negative controls or those inoculated with B. dothidea. Inoculations of shoots with B. dothidea produced similar symptoms as those of B. rhodina. Shoots that served as negative controls did not develop symptoms. Because panicle and shoot blight of pistachio caused by B. dothidea has developed to epidemic levels in commercial pistachio orchards and is of concern to the pistachio industry in California, it would be of interest to monitor how much shoot blight caused by B. rhodina would eventually develop over the years in commercial pistachio orchards. A survey was initiated in 2002 to determine how widespread B. rhodina is in California pistachios. To our knowledge, this is the first report worldwide of B. rhodina causing shoot blight of pistachio. Reference: (1) T. Michailides. Panicle and shoot blight. Page 68 in: Compendium of Nut Crop Diseases in Temperate Zones. B. L. Teviotdale, T. J. Michailides, and J. W. Pscheidt, eds. American Phytopathological Society, St. Paul, MN 2002.

Dynamics and Pattern of Latent Infection Caused by Botryosphaeria dothidea on Pistachio Buds.

A study was conducted in four commercial pistachio orchards to monitor the presence and pattern of external contamination and latent infections of buds by Botryosphaeria dothidea between 1998 and 2000. Symptomless buds were sampled every 2 to 3 weeks and analyzed either by a washing/crushing method or by direct plating of split (half) or intact buds on lactic acid potato dextrose agar. The proportion of infected buds varied among orchards over time. Levels of latent infections were highest in February and March when they reached as much as 60% in orchards of Glenn and Yolo counties. This period corresponded to the months with the highest rainfall. Buds collected from orchards in Glenn and Yolo counties had, in general, higher incidence of infection than buds from San Joaquin and Merced counties. Buds became infected in June immediately after their formation. Infection incidence on basal segments of buds from male trees was approximately twice (19%) that of apical or middle segments (11%). Plating of split buds resulted in similar levels of incidence as the plating of intact buds. The number of B. dothidea propagules on bud surfaces varied over time and among orchards. In general, the number of propagules per bud was highest in February and March (approximately seven propagules per bud) when the rainfall amount was highest.

First Report of Aspergillus Vine Canker of Table Grapes Caused by Aspergillus niger.

A vine canker was first observed in the San Joaquin Valley, CA, in fall 1989, on exceptionally vigorous 1-year-old cv. Redglobe vines (Vitis vinifera) when vines were trained up the stakes. Since 1989, the same canker symptoms have been observed in Tulare, Kern, Fresno, and Riverside (Coachella Valley, CA) counties on cv. Redglobe, Crimson Seedless, Chardonnay, and Grenache vines. In affected vineyards, the disease resulted in the retraining of 2 to 6.1% of vines the following spring, using a shoot originating from below the canker. In a sample of 54 infected vines collected in 1997, 65% of cankers were found at the branching (crotch) of the vine, 24% along the shoot, or both (11%). All infections started through wounds caused by removing lateral shoots or leaves when the vine was topped to form cordons or possibly through growth cracks that occur on rapidly growing 1-year-old shoots. The first symptoms usually appear in August as red pinhead-size drops of sap on the surface of discolored tissue. By October to November, the canopies of vines girdled by the canker prematurely display fall colors and are very distinct from healthy vines. The trunk is slightly swollen and spongy where the canker occurs. Internal canker tissue is discolored and dead. Black spores are abundant within the canker, on the surface of the canker, or both. Callous tissue is often associated with the canker as the vine attempts to repair the damage with new tissue. Canker length can range from 3.5 to 26.5 cm (average 7.0 cm) and can affect the shoot's cross section from 0.4 cm to completely girdling the shoot (up to 9.0 cm in circumference). Isolations from cankers or black sporulation inside the canker on acidified potato dextrose agar (APDA) consistently yielded Aspergillus niger van Tiegh. Six well-matured current-season canes of cv. Redglobe in an experimental vineyard at Kearney Agricultural Center were inoculated by inserting a 7-mm plug of mycelium from actively growing cultures on APDA in a cut made with a 7-mm cork borer or by brushing spores of the culture over the surface of six canes wounded with a sterile razor. Six canes were inoculated with a 7-mm plug of APDA and used as noninoculated controls. Inoculated sites were sealed with Parafilm to avoid dehydration. Inoculation of grapevines with A. niger resulted in cankers similar to those observed in commercial vineyards 5 months after inoculation. Cankers ranged from 2.4 to 4.2 cm for mycelial-plug inoculation (100% of canes infected) and 2.3 to 7.3 cm for spore-brushing inoculation (67% infected). Noninoculated control canes were not infected. In another experiment, inoculation of 10 canes each with A. niger on 17 May, 10 June, 2 July, 21 July, and 16 August resulted in 50, 60, 90, 90, and 100% canker formation, respectively, 5 to 8 months after inoculation, suggesting summer inoculations were more effective than spring inoculations. Reisolation from infected canes on APDA revealed A. niger. Aspergillus species in section Nigri have been reported to be among the pathogens involved in the bunch rot complex (1,2), but to our knowledge, this is the first report of A. niger causing a serious canker of vigorously growing grape vines. References: (1) W. B. Hewitt. Berry rots and raisin molds. Pages 26-28 in: Compendium of Grape Diseases. R. C. Pearson and A. C. Gohen, eds. The American Phytopathological Society, St. Paul, MN, 1994. (2) W. R. Jarvis and J. A. Traquair. Plant Dis. 68:718, 1984.