Charcoal rot, caused by Macrophomina phaseolina, is one of the important fungal diseases of strawberry in California. Macrophomina phaseolina is a soilborne fungus and has a wide host range, including alfalfa, cabbage, corn, pepper, and potato, some of which are cultivated in the strawberry production areas in California. The fungus infects the vascular system of the plant roots, obstructing the nutrient and water supply and ultimately resulting in stunted growth, wilting, and death of the plant. The fungus survives in the soil and infected plant debris as microsclerotia (resting structures made of hyphal bodies) and can persist for up to three years. Microsclerotia germinate and penetrate the root system to initiate infection. Plants are more vulnerable to fungal infection when they are experiencing environmental (extreme weather or drought conditions) and physiological (heavy fruit bearing) stress.
Soil fumigation is the primary management option for addressing charcoal rot in strawberry. Additionally, crop rotation with broccoli can reduce the risk of charcoal rot due to glucosinolates and isothiocyanates in broccoli crop residue that have fungicidal properties. Beneficial microorganisms such as Bacillus spp. and Trichoderma spp. are also considered, especially in organic strawberries, to antagonize M. phaseolina and other soilborne pathogens and provide some protection. The role of beneficial microbes in disease management or improving crop growth and health is gaining popularity in the recent years with the commercial availability of biofungicide, biostimulant, and soil amendment products. In a couple of recent strawberry field studies in Santa Maria, some of the beneficial microbial products improved fruit yield or crop health. These treatments can be administered by inoculating the transplants prior to planting, immediately after planting or periodically applying to the plants and or the soil. Adding beneficial microbes can help improve the soil microbiome especially after chemical or bio-fumigation and anaerobic soil disinfestation.
Similar to the benefits of traditionally used bacteria (e.g., Bacillus spp. and Pseudomonas spp.) and fungi (e.g., Glomus spp. and Trichoderma spp.), studies with entomopathogenic fungi (EPF) such as Beauveria bassiana, Isaria fumosorosea, and Metarhizium spp. also demonstrated their role in improving water and nutrient absorption or antagonizing plant pathogens. The advantage of EPF is that they are already used for arthropod pest management in multiple crops, including strawberry; now, there are the additional benefits of promoting crop growth and antagonizing plant pathogens. In light of some promising recent studies exploring these roles, a study was conducted using potted strawberry plants to evaluate the efficacy of two California isolates of Beauveria bassiana and Metarhizium anisopliae s.l. and their application strategies against M. phaseolina.
Methodology
About six-week old strawberry plants (cultivar Albion) from a strawberry field were transplanted into 1.6-gallon pots with Miracle-Gro All Purpose Garden Soil (0.09-0.05-0.07 N-P-K) and maintained in an outdoor environment. They were regularly watered, and their health was monitored for about five months prior to the commencement of the study. Conidial suspensions of the California isolates of B. bassiana and M. anisopliae s.l. were applied one week before, after, or at the time of applying microsclerotia of M. phaseolina to the potting mix. The following treatments were evaluated in the study:
Untreated control
Soil inoculated with phaseolina
Soil inoculated with bassianaone week prior to M. phaseolina inoculation
Soil inoculated with anisopliae s.l. one week prior to M. phaseolina inoculation
Soil inoculated with bassianaat the time of M. phaseolina inoculation
Soil inoculated with anisopliae s.l. at the time of M. phaseolina inoculation
Soil inoculated with bassianaone week after to M. phaseolina inoculation
Soil inoculated with anisopliae s.l. one week after to M. phaseolina inoculation
EPF were applied as 1X1010 viable conidia in 100 ml of 0.01 percent Dyne-Amic (surfactant) solution distributed around the base of the plant. To apply M. phaseolina, 5 grams of infested cornmeal-sand inoculum containing 2,500 CFU/gram was added to four 5 cm deep holes around the base of the plant. Each treatment had six pots each planted with a single strawberry plant representing a replication. Treatments were randomly arranged within each replication. The study was repeated a few days after the initiation of the first experiment.
Plant health was monitored starting from the first week after the M. phaseolina inoculation and continued for seven weeks. Plant health was rated on a scale of 0 to 5 where 0=dead and 5=very healthy and the rest of the ratings in between depending on the extent of wilting. Data from both experiments were combined and analyzed by ANOVA using Statistix software and significant means were separated using LSD test. The influence of EPF treatments applied at different times as well as the combined effect of different applications within each fungus were compared for seven weeks. Ratings for some plants that were scorched from hot summer temperatures and died abruptly were removed from the analyses.
Results
Untreated control plants maintained good health throughout the observation period varying between the rating of 4.3 and 4.9. In general, plant health declined considerably from the 5th week after M. phaseolina inoculation. Plant health appeared to be slightly better in plants treated with EPF, but there was no statistically significant difference in any except one instance. Plants treated with M. anisopliae one week prior to the application of M. phaseolina had a rating of 3.0 compared to 1.6 rating of plants inoculated with M. phaseolina alone.
When data from different treatments for each EPF were compared, both B. bassiana and M. anisopliae s.l. appeared to reduce the wilting, but the plant health rating was not significantly different from the M. phaseolina treatment without EPF.
This is the first report of the impact of EPF on M. phaseolina with some promise. The current study evaluated a single application of EPF. Additional studies under more uniform environmental conditions and with more treatment options would be useful to improve our understanding of EPF antagonizing M. phaseolina. When growers use EPF for controlling arthropod pests, they could count on additional benefits against diseases or improving general plant health.
Acknowledgements: We thank Dr. Kelly Ivors (previously at Cal Poly San Luis Obispo) for the pathogen inoculum and Dr. Stefan Jaronski, USDA-ARS, Sidney, MT for multiplying the entomopathogenic fungal inocula.
Dara, S. K. and D. Peck. 2017. Evaluating beneficial microbe-based products for their impact on strawberry plant growth, health, and fruit yield. UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=25122
Dara, S. K. and D. Peck. 2018. Evaluation of additive, soil amendment, and biostimulant products in Santa Maria strawberry. CAPCA Adviser, 21(5): 44-50.
Dara, S. K., S.S.R. Dara, and S. S. Dara. 2017. Impact of entomopathogenic fungi on the growth, development, and health of cabbage growing under water stress. Amer. J. Plant Sci. 8: 1224-1233. http://file.scirp.org/pdf/AJPS_2017051714172937.pdf
Dara, S. K., S. S. Dara, S.S.R. Dara, and T. Anderson. 2016. First report of three entomopathogenic fungi offering protection against the plant pathogen, Fusarium oxysporum f.sp. vasinfectum. UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22199
Koike, S. T., G. T. Browne, and T. R. Gordon. 2013. UC IPM pest management guidelines: Strawberry diseases. UC ANR Publication 3468. http://ipm.ucanr.edu/PMG/r734101511.html
Vasebi, Y., N. Safaie, and A. Alizadeh. 2013. Biological control of soybean charcoal root rot disease using bacterial and fungal antagonists in vitro and greenhouse condition. J. Crop Prot. 2(2): 139-150.
Southern blight, caused by the fungus Sclerotium rolfsii, is a destructive crown rot disease that rapidly kills tomato plants. The fungus is favored by high temperatures (over 86°F), high soil moisture, dense canopies, and frequent irrigation. Southern blight survives in soil as hardened structures called sclerotia for at least five years. Each infected plant can produce tens of thousands of sclerotia and then become more widely distributed in a field with each successive field operation. Although this disease may initially only affect a few plants in the field, southern blight can be serious enough to cause significant yield loss within a season or two. With a host range of over 500 plants, this fungus can easily persist from year to year in infected crop debris.
Southern Blight Identification
Southern blight misdiagnosis is likely if it occurs in an area where it has not historically been an issue, like the Sacramento Valley. It can be easily confused with other crown rotting diseases, like Fusarium crown rot. Severely affected plants can have vascular discoloration, which may be confused with Fusarium wilt. Accurate diagnosis is critical to effective control. You can distinguish southern blight in the field based on the following diagnostic traits, one or more of which may be present. Diagnosis requires looking at the soil around the crown of the plant, in addition to the plant itself.
Small tan to reddish brown sclerotia form at the base of the plant and/or in the soil around the plant.
White fungal mycelium (thread-like strands) growing INTO the soil. No other fungus will grow extensively in the soil (Figure 1).
White fan like mycelium (thread-like) growing on the crown/affected tissues.
Plants go from healthy to dead in less than a week—much faster than most crown rots (Figure 2).
Circular disease patches. From a distance, they look like bands of dead plants.
Figure 1: Fungal mycelium growing into the soil.Figure 2 (right): Rapid plant collapse and death caused by southern blight. Photo credits: C. Swett.
If none of these characteristics are present, the best way to diagnose the disease is to put infected tissue in a plastic bag on a moist paper towel and leave at room temperature for one to two weeks. The southern blight fungus will produce distinct fan like growth within about 5-7 days (Figure 3). After about 5-14 days, it will make round white balls that turn into amber colored sclerotia (Figure 4). Both the fan growth and the sclerotia are unique to this fungus.
Figure 3: Distinct fan-like growth of southern blight.Figure 4: Sclerotia developing and turning from white to amber-colored spheres. Photo credits: C. Swett.
Southern Blight Management
Soil Moisture
Maintaining a dry surface may help reduce losses if the fungus is detected in your field. The one advantage of drip irrigation is that the soil surface can more easily be kept dry, which inhibits infection by Sclerotium rolfsii. Avoid alternating wet and dry periods—wet followed by dry episodes can be particularly conducive to disease development.
Crop Rotation
If you have detected southern blight in your field, one of the best things you can do the following year is to plant a narrow canopy crop that you can effectively manage with fungicides to prevent sclerotia from increasing.
Rotations with non-host crops are limited because of the wide host range of the pathogen. Poor-host crops such as corn and small grains (wheat, millet, oats) can help to reduce sclerotia levels in the field.Most if not all of these crops can become infected by the fungus, but either they are not good hosts and/or the environmental conditions during the growing season are not favorable for pathogen growth. For instance, wheat can be a host, but it’s typically too cold for fungal growth during the time that wheat is grown. On the other hand, rotation with highly susceptible crops such as legumes (beans, peas and hairy vetch) can greatly increase soil infestation levels. Mustard cover crops can suppress southern blight, and may be useful for organic producers, where fumigation is not an option.
Soil Treatment
Deep plowing will bury the sclerotia and prevent it from attacking plants at the soil line. Sclerotia deeper than six inches are usually parasitized by other microbes and killed over time. Of course, plowing is not an option for fields where buried drip irrigation systems are already installed.
Sclerotia near the surface of the soil can be killed when exposed to high temperatures (105-120°F) for two to four weeks during the summer months. Solarization alone is not generally considered a viable management strategy, but when soils were solarized before the addition of biological control or a fungicide, disease was reduced by 70-100 percent compared to the same biological or chemical treatment without solarization. Make sure to prepare the soil for planting before solarizing, since cultivation and the incorporation of amendments can bring buried sclerotia back to the upper soil layers.
Monitoring Southern Blight Prevalence in Colusa County
Southern blight is not usually considered to be a widespread problem in California—major impacts are generally limited to Kern County. However, in 2017, late spring rains in the Sacramento Valley led to later planting dates, followed by record high temperatures, even consecutive nights where the temperature remained above 70°F. The combination of late planting dates and record high temperatures in 2017 created unusually favorable conditions for the pathogen in northern California. In 2018, we conducted a project funded by the California Tomato Research Institute to monitor southern blight prevalence in Colusa County, which had five fields with positive southern blight diagnoses in 2017. The objective of this research was to quantify southern blight spread and impact in annual rotations in the region. Sarah Light-Area Agronomy Advisor was involved in this project in addition to the authors.
We sampled soil from eight fields, five of which were confirmed to have southern blight in 2016 or 2017, the other three thought to have southern blight based on grower and pest control adviser experience and observations. Six of the fields were in tomato in 2017, one was in tomato in 2016 and wheat in 2017, and one was planted with canary bean in 2017. We sampled the soil in May 2018 to get baseline data on early season southern blight sclerotia levels. The rotational crops in 2018 included sunflower and corn. Sunflower fields were checked twice monthly for southern blight symptoms once temperatures were over 90°F for seven consecutive days because sunflower is a known host of southern blight. Tomato fields near or adjacent to the monitored fields were also checked for southern blight symptoms twice a month. Soil was collected from the same spots in the fields in August/September 2018 to determine if there were any changes to southern blight sclerotia levels in the field. Cassandra Swett’s lab analyzed the soil samples using the methanol method (Rodriguez-Kabana et al 1980). Methanol kills most microbes, but not southern blight. Trays of soil were sealed in plastic bags so the moisture could stimulate germination of the southern blight sclerotia. The germination of sclerotia was evaluated at 3 and 7 days (Figure 5).
Figure 5: Southern blight sclerotia germination in soil samples. Note white thread-like mycelial growth.
Sclerotia were recovered from three fields in May 2018, possibly five fields but identification was unclear for two of the fields. The confidence level for identifying southern blight was whether the germinated growth in the trays produced sclerotia. Germination was observed in the two samples where identification was unclear, but no sclerotia were produced from these colonies. The end-of-season samples from August and September 2018 contained much higher volumes of soil than the May samples, and we recovered sclerotia from seven of the eight fields. For total number of sclerotia, five fields had increased sclerotia levels, two fields decreased, and one field had the same number of sclerotia in both the May and September samples. Southern blight increased in all three of the sunflower fields, which was expected since sunflower is a host crop. Corn fields were spread between increases, decreases, and no changes. It is worth noting though, that the only fields with decreased levels were corn fields. Also, fields where no sclerotia were recovered may contain southern blight that was not captured among our samples.
Sunflower is not recommended as a rotational crop because it is a southern blight host. Corn is likely a better choice for rotation.
We were able to recover southern blight sclerotia in fields throughout Colusa County and demonstrate southern blight increases over the growing season with certain rotational crops. Unlike 2017, southern blight was not a large issue for tomato growers in 2018. Because southern blight requires specific conditions for development to occur, it remains a disease that is a problem in the Sacramento Valley only when environmental conditions are ideal for development, especially for certain fields. Currently in 2019, due to late spring rains and high temperatures, we have identified southern blight in the Sacramento Valley from a few tomato and vineseed fields.
We would like to thank our grower and pest control adviser cooperators on this project. We would also like to thank the California Tomato Research Institute for funding this project.
References
Rodriguez-Kabana, R., Beute, M. K., & Backman, P. A. 1980. A method for estimating numbers of viable sclerotia of Sclerotium rolfsii in soil. Phytopathology, 70(9), 917-919.
Figure 1. The biological agent, Aspergillus flavus strain AF36, producing large sclerotia (black, spherical structures in the plate).
It was in 1991, when I received a call by the President of the former California Pistachio Commission, Karen Reinecke, asking if there was a way to get involved as a technical member of the newly then established Aflatoxin Elimination Workgroup. The goal of this Workgroup was to evaluate proposals submitted by the United States Department of Agriculture (USDA) and University researchers to the USDA Special Fund to participate toward research to eliminate the problem of aflatoxin contamination by the year 2000. We are now in the second half of 2019 and aflatoxin was not only eliminated by 2000, but indeed contamination created major problems in some years in both pistachio and almond.
Our first proposal submitted in 1992 to the USDA/ Aflatoxin Elimination Technical Committee. It was funded and research started after hiring postdoc associate Dr. Mark Doster, a University of California (UC) Davis graduate with postdoc research at the University of Cornell. Mark an expert in fungal pathology and practical plant pathology was immersed quickly in this research. We were also supported then by the California Pistachio Industry which supplemented funding to intensify the research to reduce aflatoxins and find solutions for the growers. At the same time other researchers from UC Davis focused on aflatoxin research to reduce it in almond and walnut. Later on, we expanded our aflatoxin management research and included almonds and figs.
Aflatoxins are toxic compounds produced mainly by certain molds called Aspergillus flavus and A. parasiticus when these molds grow on various susceptible crops. These molds produce toxins that are considered toxigenic. The toxins produced are called aflatoxins which are considered as the most potent naturally-produced carcinogenic compounds causing liver cancer and in acute situations deaths. Strains of Aspergillus flavus that do not produce toxin are called atoxigenic, and they act as biological control agents. When they are applied on the orchard floor displace the toxin-producing mold strains, and reduce the potential for aflatoxin contamination in various crops.
There are four major aflatoxin types: the B1 and B2 produced by both the above mentioned fungi and G1 and G2 produced only by A. parasiticus. B1 is the most toxic among the four aflatoxin types. Because of this high toxicity these compounds are regulated strictly by various governments, and in fact, the B1 is regulated separately. For instance, in the USA the tolerance for B1 is 10 ppb (parts per billion) and for all the aflatoxins (total) is 15 ppb. The European Union (EU) has even stricter tolerances, i.e., 8 ppb for B1 and 10 ppb for total aflatoxins. One can judge the seriousness of aflatoxin contamination not only from the very strict tolerances but also from the losses and additional costs associated with the re-sorting and losses associated with the dumping the contaminated product. It should be noted that when shipments exceed the threshold, the consignments are rejected and must either be reconditioned or destroyed.
Below is a historical summary how this technology developed to help our California nut crop industries and the fig industry.
In the first eight years we focused on cultural practices that affect the predisposition of the pistachio crop to aflatoxin contamination and also find out whether contaminated nuts show special characteristics that can be used to sort out these nuts at the processing plant.
Below is a list of the findings from those studies led by Drs. Doster and Michailides:
We confirmed that early split nuts (ES) contained large amounts of aflatoxins and we named these nuts the “Achilles Heel” for aflatoxin contamination.
ES by themselves explained 84 percent of the aflatoxin contamination in the samples we analyzed.
When the ES were combined with the navel orangeworm (NOW) damaged nuts explained 99 percent of the aflatoxin contamination in the samples we analyzed.
We reduced the incidence of ES by providing the trees with sufficient water during early season (May) when it is the critical time for the full size development of nut shell (water stress of trees during May leads to higher incidence of ES).
We compared the incidence of ES on Kerman under the influence of four rootstocks: UCB1 and Pioneer Gold I resulted in significantly lower ES incidence than Pistacia atlantica and Pioneer Gold II rootstocks.
In my first Aflatoxin Elimination Committee (AEC) Meeting in 1991, in Peoria, Illinois, I learned for the first time that some strains of Aspergillus flavus do not produce aflatoxins and some USDA researchers reported that a very large portion of the A. flavus population consisted of strains that do not produce any aflatoxin, called atoxigenic strains. They started using these atoxigenics as candidates for biological control of aflatoxigenic fungi. It was also discovered that the proportion of strains that produced aflatoxin included strains that produced variable amounts of aflatoxins. USDA researchers started first working with atoxigenic strains to be used as biocontrol agents to reduce aflatoxins in the various crops. One of this strains was initially selected in Arizona from samples taken from cotton fields, and the Cotton Research Council of Arizona that supported financially this research were able to get AF36 registered for use in cotton and corn in 2008. Meanwhile starting in 2002, we discovered the same strain was the most commonly encountered strain among the other atoxigenic strains in pistachio, almond, and fig orchards in California, and immediately included this strain in our aflatoxin biocontrol studies. With multiyear support of USDA funding and funding by the pistachio and fig industries in California, we were able to show that this strain is among the most common atoxigenic strains occurring in California nut crop and fig orchards, and indeed it can be found at much higher incidence in comparison with all other atoxigenic strains. For instance, during these studies 15 different groups of atoxigenic strains were determined, and each one was at a rate of less than one percent, while the AF36 atoxigenic strain (Figure 1) was found in an average of five to eight percent depending on the field and the type of the crop, and in some instances up to 12 percent of populations of the atoxigenic strains
Initially, the studies were confined in small experiments in replicated micro-plots where we showed that when the AF36 was applied once preseason, it persisted well until the next application in the following year and displaced the toxigenic A. flavus strains at a rate of 90 to 95 percent displacement. These results, the fact that this strain was native to California orchards, and it was the most common atoxigenic strain and the toxicological data developed by the USDA in Arizona were sufficient to submit to the Environmental Protection Agency (EPA) to request an experimental use permit (EUP) to test the strain commercially on a larger acreage without the requirement to follow crop destruct requirements as needed with application of experimental compounds. The EUP was approved in 2008 and 3,000 acres of pistachios were treated with the commercial product of Aspergillus flavus AF36 strain produced in the Cotton Council of Arizona facility in Phoenix, Arizona. Also, 3,000 acres of pistachio close to the AF36-treated orchards were used as untreated controls. All this acreage for the EUP was provided by the former Paramount Farming Company (now Wonderful Orchards Company). At harvest the treated and the untreated fields were sampled separately and the samples, specifically called “library samples”, were analyzed for aflatoxins. For four years we showed a significant reduction of aflatoxins (Figure 2A). The average of this reduction for the four years of the EUP was close to 40 percent. When library samples of reshakes were analyzed for aflatoxins, this reduction in one year reached to 85 percent (Figure 2B). These commercial efficacy data were sufficient to obtain registration of AF36 for use in pistachio in the states of California, Arizona, Texas, and New Mexico. After the registration of AF36 on pistachio, the Almond Board of California and the California Fig Institute (the latter has funded research during the early stages of our aflatoxin research) became interested in completing any additional research so that the AF36’s registration is expanded to include almonds and figs.
Figure 2. Reduction of aflatoxin contamination (left, main crop; right, reshakes) during the years of experimental use permit (2008 to 2011) after application of AF36 strain in commercial orchards.
It took 5 years of additional research (funded by the Production Research and Food Safety and Quality Committees of the Almond Board of California) to provide the USEPA and the California of Pesticide Regulations Department the additional data for the registration of AF36 for use on almond and figs. Meanwhile because the manufacturer of the commercial product changed the initial wheat carrier of the AF36 strain to sorghum (Figure 3) the product was registered as AF36 Prevail® in January 2017 and included all, pistachio, almond, and fig. Although the pistachio industry adapted the use of AF36 widely and from 75,000 acres treated in 2012 reached to up to 200,000 acres in 2018, the almond industry was a little hesitant in widely adopting this new technology, despite the fact that there was good efficacy in reducing aflatoxin contamination in pistachio orchards. We expect to see better efficacy when all pistachio, almond and fig orchards are treated on an areawide basis because the spores of the biocontrol can spread from field to field easily with even slight windy conditions and dust.
Figure 3. The sorghum career of the AF36 Prevail® commercial product.
Description of the Biocontrol Product
Initially the AF36 product used sterilized wheat as the carrier. Sterilized wheat seed was inoculated and incubated under certain favorable conditions for the strain to invade and grow in the entire wheat seed. More recently and after additional studies in Arizona it was determined that sorghum, which has a lower cost was as good carrier as the wheat (even better under conditions in cotton and corn fields) and the manufacturer replaced the wheat with sorghum and at the same time changed the method of inoculations. The sorghum seed now is coated with a mix of a polymer and spores of AF36 instead of waiting for the seed to be colonized by the atoxigenic mold. This was done in order to satisfy the increased demand for tons and tons of inoculum since the rate is 10 lbs per acre. Studies in California under orchard conditions indicated that the sporulation on the sorghum seed is delayed, although by the end of a week both sorghum and wheat showed similar sporulation rates. The time of production of spores and the rate of sporulation are very critical for the successful use of this biocontrol agent. It is a numbers game: we want to overload the soil with atoxigenic spores and displace the spores of the toxigenic molds. That is the way this biological control approach works in the field (see challenges at the end of this article relevant to sporulation).
Proper Way for Ground Application:
For detailed information please read the label of the product:
Michailides emphasized that as far as we know up to now there are four critical application factors that need to be taken into account for AF36 Prevail to be successful in the orchard: a) the timing of application; b) the rate (amount) per acre; c) the proper placement on the orchard floor; and d) the proper irrigation before and after the application.
Timing for pistachio, almond, and fig: ApplyAspergillus flavus AF36 to the surface of the soil under the plant canopy with a granular applicator and do not cover the AF36—colonized grain with soil. For pistachios apply the product from late May through July, for almonds from late May to early July, and for figs from early May to early June. Specifically in almonds, application should be timed around hull split. If you know when to expect hull split, you should time application about one to two weeks before. You want to have the max sporulation of the biocontrol during the hull split stage of the nuts.
The rate (amount): The proper application rate is 10 pounds per acre. A single application should be made each year.
Proper placement: AF36 Prevail should be applied within the berm area of the orchard, not at row middles, so that it will be reached by the irrigation system and minimize delivery to areas that do not get wet.
Proper irrigation: Irrigation is required directly after application. Irrigation within three days after application of Aspergillus flavus AF36 will improve efficacy. The AF36 product will not sporulate without moisture and can fail if there is too much moistureAim for soil moisture levels around 13-18 percent. Proper placement within the berm, close to the irrigation system, will ensure it is successfully activated.
“Conditioning” of the orchard floor before application: This is practice that some growers have figured out on their own. Pre-irrigating and then about two days later apply the AF36 inoculum and then apply irrigation as in (d) above. Although Michailides and his crew do not have any data to support this practice, they strongly believe the practice of pre-irrigation will speed up the sporulation of the product since the rehydration can start as soon as the product comes in contact with the pre-wetted soil.
A 45 Percent Reduction of Aflatoxins is a Reality Now
Dr. Themis Michailides, plant pathologist at the UC Davis/Kearney Agricultural Research and Extension Center and former member of the National Aflatoxin Elimination Technical Committee, and Dr. Mark Doster have devoted decades to studying aflatoxins and their work has been instrumental in development and registration of AF36 in pistachio, almond, and fig.
Until now, Michailides notes, tree nut and fig growers had no direct way to combat aflatoxin. Instead contamination has been managed primarily through preventing navel orangeworm damage. While as noted below, effective orangeworm management is still very critical and essential in reducing crop damage, AF36 offers an additional tool that has a direct impact on reducing harmful toxigenic Aspergillus mold strains and the aflatoxin they produce.
Recent Challenges with the use of Aspergillus flavus AF36
AF36 Prevail can result in more than 80 percent reduction of aflatoxin contaminated cotton and corn, but here in California, only once we reached an 85 percent reduction (Figure 3) and this was only in the second harvest (reshakes) pistachios, which have higher risk for NOW infestation and aflatoxin contamination than the pistachios of the main (first) harvest. Results of analyses of a large number of library samples obtained from treated and untreated fields of Wonderful Orchards Company, resulted in a significant reduction of up to 45 percent in aflatoxin contamination. Michailides’ lab crew is trying to explain why the efficacy of AF36 in reducing aflatoxin cannot match the one reached in cotton fields in Arizona and corn fields in Texas. The following challenges may explain partially these disadvantages of the AF36 product in California: a) inadequate soil moisture and temperatures; b) not correct timing of application; c) delayed harvest, and d) inefficient control of NOW, both contributing to increased aflatoxin contamination; e) arthropod pests of career seed; and f) other predators (rodents, birds, etc.). Recent research projects are focused on addressing all these challenges. One new development is that a new product (Afla-Guard®, manufactured by Syngenta Company) that is registered to reduce aflatoxin contamination in peanuts and corn was introduced in California for experimentation and gathering data to support registration for use in pistachio, almond and fig. If successful, this will be the second product registered in the USA which represents a different from the AF36 atoxigenic strain of Aspergillus flavus and the carrier is barley (Figure 4). Studies done by Drs. Jaime and Michailides (Kearney Agriculture Research and Extension Center) in 2017 and 2018 showed that this product sporulated faster, better, and under lower temperatures and lower soil moisture content than the Aspergillus flavus AF36 strain. Registration of this product in California is anticipated in 2020, so that growers may then have a second tool to choose to combat aflatoxin contamination in 2020.
Figure 4. Another biocontrol product (Afla-Guard®) not registered yet, using barley as the career.
NOW Management is Still Essential
Aflatoxin contamination becomes a major problem in years when damage by navel orangeworm is higher than the standard low level. Relatively studies by Drs. Michailides and Palumbo (ARS (Agricultural Research Service), USDA, Albany, CA) showed that NOW moths are heavily contaminated with spores of aflatoxigenic fungi as soon as they emerge from mummies in early spring. Also as the damage on nuts increases so is the incidence and the amounts of aflatoxins (Figure 6). Therefore, it is essential to keep up with navel orangeworm pest management practices. Growers should use all the available tools for reducing damage by NOW to supplement the mummy sanitation, which should be the first step towards aflatoxin reduction. Reduction of NOW damage can also be achieved by timely harvest, in season insecticide sprays, and winter mammy shake.
Figure 5. Correlation of the sites of feeding damage of almonds by NOW larvae and amounts of aflatoxin contamination.
Where to Find the Product, Learn More, and Application Services
To learn more about AF36 and its application, watch two short videos produced by California Pistachio Research Board. Although they were filmed in a pistachio orchard, the information is useful and accurate for almond and fig growers.
Western Milling is the distributor for AF36 in California. Growers can contact Jeff Chedester, seed business manager, at (559) 302-2593; and Agri Systems, Inc., c/o Brendan Brooks, at 559-665-2100 for product information and application. Distributors for the second product will be provided as soon as it is registered in California.
Acknowledgments:
The author thanks all his collaborators for the dedicated research, the California Pistachio Industry (California Pistachio Research Board), the Almond Board of California, and the California Fig Institute for their continuous financial support of these studies. We also thank the USDA the initial seed grants provided by the Aflatoxin Elimination Technical Committee, and California Department of Food and Agriculture (CDFA) (Grant SCB16054). Special thanks go to Wonderful Orchards for their continuous support of this research by allowing using their orchards for various experiments; also we thank Keenan, Sutton, and Nichols Farms for their support as well. In addition, we appreciate very much the support by Syngenta in 2018 and 2019.
Good quality water is extremely important for agriculture throughout the world. However, due to reduced availability of water and increasing food demands, future use of degraded waters is evident. One of the major concerns of utilizing degraded waters for irrigation is their high salt concentration.
Salinity is one of the main abiotic stresses faced by the agriculture industry. Modest increase of soil salinity level impacts both plant growth and yield by causing several physiological and biochemical changes. Based on salt tolerance level plants are classified broadly in two groups: halophytes and glycophytes. The halophytes have special mechanisms to tolerate high concentrations of salts and therefore can grow in saline environments. The majority of plants (including almonds) are glycophytes and cannot tolerate high salt concentrations and so grow in soil containing low salts. However, among glycophytes, salt tolerance level varies tremendously not only at the species level but also at the variety level within a species. This variation is directly dependent on the functional status of various molecular components that play critical roles to protect plant during salt stress.
In the initial stages of salinity exposure, a plant faces osmotic stress, resulting in ion imbalance in cells, membrane disintegration and reduced photosynthesis. In addition, osmotic stress in the root sends a signal throughout the plant causing reprograming of physiological and molecular activities to initiate defense response against salinity stress. Slowly ionic stress develops, leading to accumulation of Na+ (sodium ions) and Cl- (chloride ions) in plant tissues. High ion concentrations are not only toxic but also interfere with absorption of essential nutrients by a plant. High Na+ and Cl- levels interfere in many molecular, biochemical, metabolic and physiological processes which could also lead to unnatural senescence and cell death. For plant species that are moderately tolerant to salinity, osmotic stress may play an important role. However, for species sensitive to salinity such as almonds, low salt concentration is able to impose ionic stress that may reach to an intolerable level, whereas it may not generate osmotic stress critical for plant growth. Hence, when studying salinity stress in almonds, it is important to focus on responses related to ionic stress and the exposure to salinity should be gradual to avoid any osmotic shock. This also mimics field conditions in an almond orchard, as during spring salt moves to subsurface layers due to the rain and slowly moves to upper layers during summer, which leads to gradual increase in root zone salinity.
Due to a continuous increase in cultivated area under almonds, farmers are forced to utilize marginal lands with low quality saline water for irrigation. In almonds, rootstock plays an important role in regulating plant growth in salinity stressed environment. Hence, the development of new almond rootstocks tolerant to salinity is highly desirable. In last two decades, several studies focusing on screening almond rootstocks for salinity tolerance have been conducted and some tolerant rootstocks have been identified. However, a comprehensive approach to screen and develop new rootstocks with enhanced salinity is missing in almonds.
Impact of Salinity on Water Relations and Photosynthesis
Water uptake by a plant is drastically affected under salinity, which leads to reduced water potential, relative water content, stomatal conductance and transpiration. As plants take nutrients with water, reduced water uptake also decreases tissue concentration of essential nutrients affecting plant growth. In addition, high salt concentrations also affect homeostasis, osmoregulation and net photosynthesis. Photosynthesis is the most critical metabolic process for almonds. In response to salinity, osmotic stress-mediated stomatal closure prevents water loss through transpiration in plants that also restricts the amount of CO2 taken in for photosynthesis. Consequently, stomatal conductance, net photosynthetic rate and amount of chlorophyll are used as physiological parameters to study salinity tolerance in different almond varieties. In a recent study where we compared several rootstocks for salinity tolerance, photosynthetic rate was found to be the most reliable parameter to assess salinity tolerance.
Tissue Ion Composition and Salinity Tolerance
Tissue Na concentration is commonly used as a guide for the salinity tolerance of a variety. However, for some plant species, tissue Na concentration is not a true indicator of salt tolerance of a variety. Never-the-less, for almonds the negative correlation between tissue Na concentration and salt tolerance holds well. Similar to Na accumulation, salt tolerant genotypes stored least amount of Cl in leaf tissue.
Figure 1. Evaluation of salinity tolerance of 16 almond rootstocks irrigated with waters of different ion compositions. Photo courtesy of Devinder Sandhu.
Genetic Control of Salinity Tolerance
In model plants, hundreds of genes have been discovered that play critical roles in salinity tolerance. Salt-stress induced signaling pathways have also been well-dissected. Molecular mechanisms of salt tolerance or salt sensitivity is largely unknown in almonds. Expression analysis of genes involved in ion transport in almond tissue showed induction of multiple genes involved in Na+ and Cl– transport under salinity treatment, suggesting importance of both Na and Cl during salinity stress. The genes involved in Na+ transport were differentially expressed during salinity stress, compared to the control. For instance, NHX1 (a vacuolar sodium/proton antiporter) and SOS3 (SALT OVERLY SENSITIVE 3 that encodes a calcium sensor) were upregulated in leaves and on the other hand HKT1 (encodes a Na transporter) was induced in roots under salinity treatment. SOS3 is involved in Na+ exclusion from roots, NHX1 plays role in sequestering Na+ in vacuole and HKT1 is critical for retrieving Na+ from xylem back into root to protect leaves from salt toxicity. Additionally, CLC-C (chloride channel C) and SLAH3 (encodes a slow-type anion channel) that are important for Cl– transport, were highly upregulated in salinity treatments in almond roots. These observations confirmed the role of multiple component traits in salt tolerance mechanism in almonds. As seen in other plants, multiple signaling pathways and various genes are expected to be involved in establishing ionic homeostasis during salt stress. Nevertheless, there are not many studies focusing on understanding the roles of organic solutes and enzymatic or non-enzymatic antioxidants in mitigating effects of salinity in almonds. Although some genes involved in ion exclusion, and ion sequestration in vacuoles have been identified in almonds, future studies are warranted to identify additional genes. In addition, different scions should also be compared for the genetic variation involved in ion homeostasis and scavenging reactive oxygen species (ROS) produced during salinity stress, which may provide some insights into sensitivity of almonds to salinity.
Contrary to studying the importance of a few genes in salinity stress at a time, an RNA-seq based approach compares global changes in gene expression between the control and the salinity treatments. In addition to targeting genes already characterized in model plants this strategy can link different pathways involved in salinity tolerance and identify specific almond genes contributing toward salt tolerance.
Can Alternate Approaches Mitigate Harmful Effects of Salinity?
Many additional strategies have been reported in plants that have been implicated to improve salt tolerance level in response to salt stress. For instance, application of certain microbes could improve salt tolerance level of plants. Arbuscular mycorrhizal fungi (AMF) are known to form symbiotic associations with many land plants that considered to be valuable to plant growth. AMF helps host plants not only by providing essential minerals but also by impeding the translocation of toxic ions like sodium. The use of AMF in multiple plant species has shown enhanced growth, development and productivity under salt stress. Although, different Prunus rootstocks have been screened for mycorrhizal colonization, direct effect of AMF in mitigating salinity still need to be established.
Application of plant growth promoting rhizobacteria (PGPR) is also known to improve salt tolerance in different plant species. However, there are no published reports describing effect of PGPR in improve salt tolerance in almonds.
Although, AMF and PGRP show a lot of promise, the potential of their application on almond rootstocks to mitigate salinity stress needs to be explored further, along with the economic feasibility of these approaches at the commercial level.
Future Perspectives
One of the main consequences of the climate change is the length and frequency of drought periods experienced in certain part of the world. California, the main almond producing region of the world, experienced a long drought period in the recent past. Drought leads to excessive groundwater pumping and use of alternative water resources with high salinity for irrigation. Based on the current trends, salinity problem is expected to intensify in next couple of decades. Currently, salinity screening is taking a backseat in almond rootstock breeding, which is expected to change in the near future. One of the approaches for the future almond breeding programs will require screening of wild genetic material for salinity tolerance. In addition to the other important rootstock traits such as high vigor, nematode resistance, disease resistance, insect resistance, drought tolerance, salinity tolerance should also take central stage during rootstock breeding.
Identification and isolation of the key almond genes involved in salinity tolerance will be critical. Functional validation of selected almond genes by complementation assay in a model plant like Arabidopsis may provide an initial proof of functional conservation of genes between these species. Characterization of genes will facilitate identification of specific mutations that are critical for salinity tolerance. The CRISPR/Cas9 system has a great potential in fixing the both type of genes that play positive or negative roles in salt tolerance in almonds. The CRISPR/Cas9 is a precise, suitable, and efficient technology that has been used for genome editing in various crops such as rice, wheat, maize and sorghum. It is important to note that CRISPR/Cas9 modified crops are not considered as genetically modified organisms (GMO).
Identification and characterization of genes regulating ion uptake, effective compartmentalization, and tissue tolerance may provide new means to develop almond varieties with enhanced salinity tolerance.
California is the leading producer of tomatoes, especially for the processing market (California Department of Food and Agriculture (CDFA), 2019). Tomato is the 8th most important commodity in California valued at $1.05. Processed tomatoes are ranked 6th among the exported commodities with a value of $813 million. While good nutrient management is necessary for optimal growth, health, and yields of any crop, certain products that contain minerals, beneficial microbes, biostimulants, and other such products are gaining popularity. These materials are expected to improve crop health and yield, impart soil or drought resistance, induce systemic resistance, or improve plant’s immune responses to pests, diseases, and other stress factors (Berg, 2009; Bakhat et al., 2018; Chandra et al., 2018; Shameer and Prasad, 2018). Maintaining optimal plant health through nutrient management is not only important for yield improvement, but it is also an important part of integrated pest management strategy as healthy plants can withstand pest and disease pressure more than weaker plants and thus reduce the need for pesticide treatments.
Experimental plots, transplanting, and treatment details. All photos courtesy of Surendra K. Dara.
Methodology
A study was initiated in the summer 2017 to evaluate the impact of various treatment programs on tomato plant health and yield. Processing tomato cultivar Rutgers was seeded on 7 June and transplanted on 18 July, 2017 using a mechanical transplanter. Monoammonium phosphate (11-52-0) was applied at 250 lb/ac as a side-dress on August 7th as a standard for all treatments. Since planting was done later in the season, crop duration and harvesting period were delayed due to the onset of fall weather. Plots were sprinkler irrigated daily or every other day for three to four hours for about two weeks after transplanting. Drip irrigation was initiated from the beginning of August for 12-14 hours each week and for a shorter period from mid October onwards.
There were five treatments in the study including the standard. Each treatment had a 38 inch wide and 300 foot long bed with a single row of tomato plants. Treatments were replicated four times and arranged in a randomized complete block design. Different materials were applied through drip using a Dosatron injector system, sprayed at the base of the plants with a handheld sprayer, or as a foliar spray using a tractor-mounted sprayer based on the following regimens.
Standard
AgSil® 21at 8.75 fl oz/ac in 100 gal of water through drip (for 30 minutes) every three weeks from 31 July to 13 November (6 times). AgSil 21 contains potassium (12.7 percent K2O) and silicon (26.5 percent SiO2) and is expected to help plants with mineral and climate stress, improve strength, and increase growth and yields.
Yeti BloomTMat 1 ml/gallon of water. Applied to the roots of the transplants one day before transplanting followed by weekly field application through the drip system from August 7th to November 13th (15 times). Yeti is marketed as a biostimulant and has a consortium of beneficial bacteria—Pseudomonas putida, Comamonas testosterone, Citrobacter freundii, and Enterobacter cloacae. Yeti Bloom is expected to enhance the soil microbial activity and helps with improved nutrient absorption.
Tech-Flo®/Tech-Spray® program contained five products that supplied a variety of macro and micro nutrients. Products were applied through drip (for 30 min) at the following rates and frequencies in 300gal of water.
Tech-Flo Omegaat 2 qrt/ac in transplant water and again on September 11th (two weeks after the first bloom).
Tech-Flo Sigmaat 2 qrt/ac on September 11th (two weeks after the first bloom).
Tech-Spray Hi-Kat 2 qrt starting at early color break on September 25th with three follow up applications every two weeks.
Innovak Global program contained four products.
ATP Transfer UPat 2 ml/liter of water sprayed over the transplants to the point of runoff just before transplanting. Three more applications were made through drip (for 30 min) on August 7th and 21st and September 4th. This product contains ECCA Carboxy® acids that promote plant metabolism and expected to impart resistance to stress factors.
Nutrisorb-Lat 40 fl oz/ac applied through drip (for 30 min) on 31 July, 14 August (vegetative growth stage), 4 and 18 September, and 2 October (bloom through fruiting). Nutrisorb-L contains poly hydroxyl carboxylic acids, which are expected to promote root growth and improve nutrient and water absorption.
Biofit®N at 2 lb/ac through drip (for 30 min) on July 31st, August 21st (three weeks after the first), and 4 September (at first bloom). Biofit contains a blend of beneficial microbes—Azotobacter chroococcum, Bacillus subtilis, megaterium, B. mycoides, and Trichoderma harzianum. This product is expected to improve the beneficial microbial activity in the soil and thus contribute to improved soil structure, root development, plant health, and ability to withstand stress factors.
Packhardat 50 fl oz/ac in 50 gal of water as a foliar spray twice during early fruit development (on September 11th and 18th) and every two weeks during the harvest period (four times from October 2nd to November13th). Contains calcium and boron that improve fruit quality and reduce postharvest issues.
A 50 foot long area was marked in the center of each plot for observations. Plant health was monitored on August 1st, 8th, and 22nd by examining each plant and rating them on a scale of 5 where 0 represented a dead plant and 5 represented a very healthy plant. Yield data were collected from October 11th to December 5th on eight harvest dates by harvesting red tomatoes from each plot. On the last harvest date, mature green tomatoes were also harvested and included in the yield evaluation. Data were analyzed using analysis of variance and Tukey’s HSD test was used for means separation.
Fig. 1. Plant health on a 0 (dead) to 5 (very healthy) rating on three observation dates.
Results and Discussion
There was no statistically significant difference (P > 0.05) in the health of the plants in August (Fig. 1) or in the overall seasonal yield (Fig. 2) among treatments. The average health rating from three observations was 3.94 for the standard, 4.03 for AgSil 21, 4.45 for Yeti Bloom, 4.38 for Tech-Flo/Spray program, and 4.35 Innovak Global program.
When the seasonal total yield per plot was compared, Yeti Bloom had 194.1 lb followed by, Innovak program (191.5 lb), AgSil 21 (187.3 lb), the standard (147.4 lb) and Tech-Flo/Spray program (136.5 lb). Due to the lack of significant differences, it is difficult to comment on the efficacy of treatments, but the yield from AgSil 21 was 27 percent more than the standard while yields from Innovak program and Yeti Bloom were about 30 percent and 32 percent higher, respectively.
Fig. 2. Seasonal total yield/plot from different treatments.Fig. 3. Percent difference in tomato yield between the standard and other treatment programs.
Studies indicate that plants can benefit from the application of certain minerals such as silicon compounds and beneficial microorganisms, in addition to optimal nutrient inputs. Silicon is considered as a beneficial nutrient, which triggers the production of plant defense mechanisms against pests and diseases (Bakhat et al., 2018). Although pest and disease conditions were not monitored in this study, silverleaf whitefly (Bamisia tabaci) infestations and mild yellowing of foliage in some plants due to unknown biotic or abiotic stress were noticed. AgSil 21 contains 26.5 percent of silica as silicon dioxide and could have helped tomato plants to withstand biotic or abiotic stress factors. Similarly, beneficial microbes also promote plant growth and health through improved nutrient and water absorption and imparting the ability to withstand stresses (Berg, 2009; Shameer and Prasad, 2018). Beneficial microbes in Yeti Bloom and Biofit®N might have helped the tomato plants in withstanding stress factors and improved nutrient absorption. Other materials applied in the Innovak program might have also provided additional nutrition and sustained microbial activity.
The scope of the study, with available resources, was to measure the impact of various treatments on tomato crop health and yield. Additional studies with soil and plant tissue analyses, monitoring pests and diseases, and their impact on yield would be useful.
Acknowledgements: Thanks to Veronica Sanchez, Neal Hudson, Sean White, and Sumanth Dara for their technical assistance and the collaborating companies for sending free samples or providing financial assistance.
References
Bakhat, H. F., B. Najma, Z. Zia, S. Abbas, H. M. Hammad, S. Fahad, M. R. Ashraf, G. M. Shah, F. Rabbani, S. Saeed. 2018. Silicon mitigates biotic stresses in crop plants: a review. Crop Protection 104: 21-34. DOI: 10.1016/j.cropro.2017.10.008.
Berg, G. 2009. Plant-microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. Appl. Microbiol. Biotechnol. 84: 11-18. DOI: 10.1007/s00253-009-2092-7.
Chandra, D., A. Barh, and I. P. Sharma. 2018. Plant growth promoting bacteria: a gateway to sustainable agriculture. In: Microbial biotechnology in environmental monitoring and cleanup. Editors: A. Sharma and P. Bhatt, IGI Global, pp. 318-338.
Shameer, S. and T.N.V.K.V. Prasad. 2018. Plant growth promoting rhizobacteria for sustainable agricultural practices with special reference to biotic and abiotic stresses. Plant Growth Regulation, pp.1-13. DOI: 10.1007/s10725-017-0365-1.
The 2017-2018 United States (U.S.) citrus crop was valued at 3.28 billion dollars with California’s citrus production accounting for 59 percent of the overall U.S. production. Much of California’s bearing acreage is devoted to orange production, however other citrus varieties of tangerine, mandarin, lemon and grapefruit are grown throughout the state. As the California citrus industry contributes to over half of the total U.S. citrus production, the identification and management of new disease threats is crucial.
Colletotrichum, a Globally Distributed Fungus
Colletotrichum constitutes a large genus of fungi which are known for having diverse ecological roles ranging from endophytes, fungi living within plant tissues and causing no known problems, to plant pathogens that can kill entire plants or portions of the plant. Colletotrichum includes some important fungal pathogens of numerous plant hosts including native and agricultural plant species occurring in tropical and subtropical regions in the world. Colletotrichum is well-known for causing various anthracnose diseases on many plants, with general anthracnose symptoms including necrotic lesions on various plant parts including stems, leaves, flowers and fruits. Although Colletotrichum is primarily described as causing anthracnose diseases, other diseases such as rots caused by Colletotrichum spp. have been documented.
Currently, over 100 species of Colletotrichum have been described and recent phylogenetic studies (which show the relationship among organisms) based on the analysis of DNA has found that at least 71 unique phylogenetic species exist within three well known ‘species’ of Colletotrichum based on traditional morphology; C. gloeosporioides, C. acutatum, and C. boninense. The large species diversity within the Colletotrichum genus highlights the importance of DNA phylogenies to accurately identify species. With respect to citrus, two species of Colletotrichum, C. gloeosporioides (Penz.) Penz. & Sacc. and C. acutatum J.H. Simmonds, have been associated with anthracnose diseases of citrus. These anthracnose diseases, which include post-harvest anthracnose, postbloom fruit drop (PFD), and key lime anthracnose (KLA) are of great economic importance as postharvest diseases. However, recent evidence is suggesting that additional species of Colletotrichum previously unknown from citrus are causing diseases of citrus globally, particularly from the C. boninense species complex.
Colletotrichum karstii You L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai (C. boninense species complex) has been increasingly reported from anthracnose symptoms of citrus worldwide and is often found to occur in association with other Colletotrichum spp., particularly C. gloeosporioides which generally predominates within citrus hosts. C. karstii has been increasingly reported from anthracnose diseases of other crops including avocado, mango, and persimmon and is considered the most common and widely distributed species of the C. boninense species complex. Although C. karstii has been reported from citrus in China, Italy, and Portugal, in the United States C. karstii has only been reported from other host species.
Figure 1. Symptoms of Colletotrichum Dieback. A) Shoot dieback symptoms on Clementine, B) Gumming symptoms on an infested shoot. C) Branch dieback symptoms on Clementine. D) Wood discoloration and canker on the wood.
Colletotrichum Symptomology
Recently, unusual disease symptoms associated with Colletotrichum spp. have been observed frequently in various citrus orchards in the San Joaquin Valley of California (Eskalen, per ob). Symptoms include leaf chlorosis, twig and shoot dieback, crown thinning, wood cankers in branches and in some cases death of young plants (Figure 1). Isolations from diseased tissues yielded typical Colletotrichum species based on colony morphology but slight differences also suggested that more than one species may be present. Historically, C. gloeosporioides sensu stricto has been the only species associated with anthracnose diseases of citrus in California.
C. karstii, a ‘New’ Species Associated With Citrus in California
Recent work by researchers at the University of California have now identified C. karstii as a new pathogen of citrus causing twig and shoot dieback with or without gumming and occasionally branch dieback and wood canker in the Central Valley of California. Pathogenicity tests on clementine mandarin also confirmed that C. karstii is a more aggressive pathogen of citrus in California compared to C. gloeosporioides based on in planta experiments (Figure 2). Based on a survey of samples collected throughout the Central Valley, this same research also found that both species are commonly isolated from symptomatic tissues and were often found co-infecting the symptomatic samples. However, the researchers also never found other known wood canker pathogen species of citrus within the Botryosphaeriaceae and Diatrypaceae from samples in which both Colletotrichum species were isolated. Unlike anthracnose which can cause twig dieback and is associated with C. gloeosporioides, this disease is associated with two species of Colletotrichum and is not limited to twig dieback alone but is also associated with shoot dieback and in some cases, woody cankers. Taken together, this confirms C. karstii as a new pathogen of citrus in California causing a disease distinct from anthracnose which is caused by C. gloeosporioides.
Figure 2. Pathogenicity of Colletotrichum spp. on ‘4B’ clementine after 15 months. Vertical lines represent standard error of the mean.
The association of C. karstii with citrus twig and shoot dieback in California represents a significant finding since C. karstii appears now to be a new pathogen of citrus in the United States. Anthracnose disease of citrus has mainly been attributed to C. gloeosporioides and C. acutatum which are considered mainly as foliar and fruit pathogens. Although symptoms of anthracnose caused by C. gloeosporioides in citrus include twig dieback, leaf drop and necrosis on fruits as a postharvest disease, a progression to shoot dieback and association with branch dieback and wood cankers has not been observed.
Figure 3. Monthly spore trap counts with temperature (°C), precipitation (mm), and relative humidity (%) for (a) Kern and (b) Tulare counties. Vertical bars represent total colony forming units (CFU) counted from each citrus orchard by month. Lines represent average monthly temperature (°C) and relative humidity (%) and total monthly precipitation (mm).
Although little is known regarding the epidemiology of C. karstii on citrus, several environmental factors are likely important for the dissemination and progression of this disease. Relative humidity and precipitation in citrus orchards in California play an important role in the epidemiology of Colletotrichum infection whereby conidia dispersed by rain and humidity are conducive to pathogen spread. Our spore trap study showed that spore trapping of Colletotrichum species occurred most frequently during the months with the highest precipitation (Figure 3), however Colletotrichum spp. were not always correlated with rainfall. Similar results were found with other spore trap studies in California. Wounding is also known to predispose plants to infection by Colletotrichum and typical agricultural practices and the environment in California citrus groves (pruning, shearing, wind/sand damage) give both of these species the opportunity to colonize citrus trees. Based on recent research, symptoms were observed during the late spring and summer months, with no new symptoms being observed into fall, winter and early spring. This suggests that young, tender tissues developing in the late spring are likely necessary for initial pathogen colonization.
Figure 3. (b).
Management Practices for Colletotrichum Dieback
Currently no strategies exist for the management of this emerging disease in citrus. Adherence to cultural practices recommended for the management of other canker and dieback pathogens should be followed. These practices include maintaining trees in good condition through appropriate irrigation regimens and proper fertilization, removal of infested branches and pruning debris during dry periods followed by immediate disposal of infested material, and sanitizing pruning equipment. Chemical management using fungicides is being investigated and these methods may become part of an integrated pest management strategy for Colletotrichum diseases of citrus in California.
Acknowledgements
Financial support for this project was provided by the Citrus Research Board (Project # 5400-152). Plants used for pathogenicity tests were kindly donated by Wonderful Citrus. We thank D. Trannam, R. Yuan, K. Sugino, Q. Douhan, and our cooperating citrus growers for assistance in the lab and field.
Figure 1. An unheaded 2nd leaf prune tree in March 2017 in Glenn County with traditionally headed trees in the background. This Glenn County grower is experimenting on their own with various tree training regimes. No photo credit necessary.
California will likely have a large prune crop in 2019 following favorable bloom conditions and lower yields in 2018. Unfortunately, in prune production with larger crops typically comes smaller fruit, of which there is currently an over-supply in the world market. High production of small fruit world-wide has come at a time when demand for small fruit from consuming nations like China, Brazil, and Russia has been in decline. California handlers have been strongly urging their growers to use shaker thinning to reduce the fruit number during spring and help deliver large, high-quality fruit at harvest.
To be successful, the prune orchard of the future is going to have to thread the needle of achieving earlier production in its life cycle and maintaining high and consistent yields in maturity, all while attaining large average fruit size each year. Prunes come into production later than many other orchard crops, one way to increase early production is to reduce or eliminate severe heading cuts during tree training. Subsequently, yield potential at orchard maturity may be increased over historical production levels in some situations by striking a new balance between spacing and rootstock vigor for increased canopy volume and higher light interception. Finally, the key mechanism for achieving more consistent yields and larger fruit size in prune production has been to thin the crop with mechanical shaking following good bloom conditions like we had this year. Shaker thinning will likely continue to be the foundation of consistent yields for future orchards.
Pruning Prunes: Greater Early Production by Avoiding Heading Cuts During Establishment
University of California (UC) research has shown that earlier and greater production can be achieved in both almonds and walnuts by reducing the severity of pruning during tree establishment. This has been done by reducing or eliminating the use of severe heading cuts (figure 1) during tree establishment. In pruning, “heading cuts” reduce the length of a limb, while “thinning cuts” completely remove the limb. Although historical orchard tree training often calls for severe heading cuts (e.g. cutting back ½ or more of the 1st year growth during the first dormant period), leaving limbs unheaded can lead to earlier fruit production.
Bill Krueger, now a UC Cooperative farm advisor emeritus, tested several pruning regimes for newly planted prune trees from 1996 through 2000. The most successful of these pruning regimes all minimized the severity of pruning. Yield and profitability were greatest in the treatment that selected three to five scaffolds at the first dormant and bent back competing limbs (nearly to, or to the point of breaking to reduce competitiveness). Competing limbs were again bent at second and third dormant, and finally limbs were left unheaded at fourth dormant. In all four years of this treatment, pruning cuts were made for selective thinning (complete limb removal) to help shape the tree into a vase-shape and eliminate competing or crossing branches. Other treatments that both yielded well and had the highest total income selected either 3 or 3-5 scaffolds and either left those scaffolds unheaded or lightly tipped in the first dormant. In the 2nd, 3rd and 4th dormant these other successful treatments had various sequences of either being unheaded (but still making thinning cuts) or leaving the trees completely unpruned. By contrast, the severe pruning treatment that had the lowest cumulative dry yield and income selected three scaffolds at the first dormant and then headed new limb growth back to 30 inches in the 1st through 4th dormant.
Joe Turkovich, Winters area grower and Chairman of the California Prune Board developed his own minimal pruning training method, which he describes as a modified version of the “long pruning” method described by Krueger and others in the UC Prune Production Manual. First used in the early 1990’s, the goal of his approach is to create an upright canopy framework with a strong interior architecture capable of bearing large crops without the need for wiring, rope, or propping. It allows for early bearing and isolates breakage to flatter, bearing side limbs. It involves the almost exclusive use of thinning cuts as opposed to heading cuts. Turkovich heads the dormant bareroot trees at 40 inches at planting to allow space for vertical separation between the future primary scaffolds. Scaffolds are selected during October of the first year of growth and left unheaded. In May of the 2nd year, the three scaffolds are lightly tipped back (approximate height nine feet) to keep them from bending out of position, and approximately a third of new growth is thinned out (in particular, removing crossing-limbs, while allowing flat fruiting wood to develop). In the dormant period between year two and three (or during the summer of year three) the only pruning that occurs is to select or promote the growth of secondary leaders (two per scaffold). Again, no heading cuts are done, and no attempt is made to “open up the tree”. Between year three & four, again, no heading cuts. Excessive side limbs are removed or tipped to prevent over-bearing and breakage and tertiary leaders are selected or promoted, two per secondary leader. In subsequent years he continues to avoid heading cuts, never topping the trees. If tree height needs to be reduced, leaders are thinned back to strong side upright limbs three to five feet below the top of the tree. On 18′ by 16′ spacing, Turkovich reports this orchard training approach has yielded approximately 0.6 tons in the 3rd leaf, 1.2 in the 4th, 3.0 in the 5th and an average of 4.5-5.5 tons/ac of high-quality fruit in the 6th year and beyond.
Whether utilizing one of the minimal pruning regimes tested by Bill Krueger or the approach utilized by Joe Turkovich, minimizing severe heading cuts during tree establishment can lead to improved early yields.
Tighter Spacing and Greater Light Interception in the Prune Orchard of the Future: Increased Canopy Volume à Increased Light Interception à Increased Yield Potential
At maturity, higher yields could be achieved in many California prune orchards by capturing more light with the choice of a more vigorous rootstock, and/or planting at a closer spacing. We all know that fruit and leaves grow on branches, and that fruit need the sugar production from neighboring leaves to grow and sweeten. Thus, one way to think about the yield potential of an orchard is how many fruit-leaf groupings (also called bearing units) are spread out over the orchard. In other words, increasing the amount of space in the orchard taken up by the orchard canopy (instead of open, unused space) will increase your yield potential per acre.
One measure of canopy size is how much light that canopy intercepts. Light that is intercepted by the leafy canopy and doesn’t reach the orchard floor is measured as midday photosynthetically active radiation (percent PAR). Work by the laboratory of Bruce Lampinen, University of California Cooperative Extension (UCCE) orchard specialist at UC Davis, has found that for every 1 percent of light that an almond orchard captures there is an average of 40 lbs/ac increased yield in all measured orchards (see figure 2). Lampinen has found this relationship between greater light capture and greater yield potential in both almond and walnut production. Light interception isn’t the only determinant of yield of course, therefore these are “potential” yields and depend on proper irrigation, fertilization, pest and disease management.
Figure 2. Measuring both almond yield (kernel lbs/ac) and midday photosynthetically active radiation (PAR) percentage have shown a direct relationship of 40 kernel lb/ac per 1 percent PAR (courtesy of Lampinen Lab, UC Davis).
Light Interception and Yield Potential in Prune Production
Although the relationship between canopy light interception and yield has not been as well studied in prune production, there does appear to be a clear relationship from the limited data available (see figure 3). Although there is substantial variation, the denser 14’ x 17’ planting in this example is achieving between 60-80 percent light interception and is clearly out yielding the wider spaced plantings that are only capturing 30-45 percent of midday light, common in many California prune orchards. The 16 foot in-row spacings of the wider plantings appear as discrete trees (they do not touch), while the 14’ x 17’ spacing have created continuous hedgerows (see figure 4). This tighter 14’ x 17’ (183 trees/acre) spacing illustrates the 6-8 dry tons/ac yield potential of prune orchards in excellent cropping years.
Figure 3. Two clusters of prune yield (dry tons/acre) versus midday light interception (%), grouped by row spacing (courtesy of E. Fichtner and F. Niederholzer).
Prune orchard spacing has historically been determined by the constraints of harvest equipment. However, some growers are instead shifting this paradigm and beginning to modify their equipment to get through tighter spacings. Many questions and potential challenges arise due to this shift in paradigm and will be addressed through experimentation by innovative growers and UC researchers.
Figure 4. Two orchards with contrasting spacing, light interception and yield potential (courtesy of E. Fichtner (L) and F. Niederholzer (R)).
For more considerations impacting spacing and light interception, including rootstock vigor and soil type, per tree costs and mechanical hedging, see: cebutte.ucanr.edu/newsletters/Prune_Notes79781.pdf
Consistent Large Fruit Size
The final component to remaining competitive with the increased production levels in Chile and Argentina, is achieving large, high-quality fruit each and every-year. The key approach to achieving consistent large fruit size is shaker thinning in years like 2019 when a high percentage of flowers set fruit (far too many for the tree to achieve a large average fruit size). Fruit thinning occurs roughly at “reference date” or when 80-90 percent of fruit have a visible “endosperm”.
Endosperm is a clear gel-like glob that can be excised with a knife point from the blossom end of the seed (see figure 5). Reference date is roughly one week after the pit tip begins to harden and timing is typically late April or early May. Although the earlier thinning is done, the great the effect will be on final fruit size at harvest, if you thin too early you can damage the tree without effectively removing fruit.
Figure 5. Extraction of the endosperm on a developing prune. Photo courtesy of M.L. Poe.
Thinning is a critical practice for several reasons. When production of small fruit is up worldwide at the same time demand for small fruit is in decline, achieving large average fruit size is an absolute imperative. Not only do small fruit have substantially less value (or even no value in some cases), they are costlier to harvest and dry. Setting a large crop of small fruit can also be a great stressor on an orchard creating a large sink for potassium demand and causing limb breakage. Finally, since not every year’s bloom creates favorable conditions for fruit set, over-cropping can set up a vicious cycle of having fewer flowers that will be blooming during an uncertain bloom period in the subsequent year. In other words, thinning enables a high flower density each year. Even if bloom conditions are poor and set is low, a low percentage of fruit set from a high number of flowers is much better than low set from only a few flowers. Mechanical thinning enables the production of higher-value fruit, avoids the drain of costly small fruit and sets up the orchard for more sustainable year-to-year production.
Three key practices for maximizing orchard productivity are utilizing reduced severity of pruning during canopy training, achieving higher yield potential by maximizing canopy light interception and consistently attaining large average fruit size through thinning. These practices may be part of what gives the California prune orchard of the future a competitive edge in the global market.
This work is made possible by the funding support of the California Prune Board. Special thanks to Mark Gilles (Sunsweet) for his input on prune orchard spacing both historically and currently. Special thanks also to Joe Turkovich who kindly provided the details of his modified version of the “long pruning” method.
Photo1. Esca tiger strip symptom on Autumn King. All photos courtesy of Gabriel Torres.
“Trunk disease” is a catch-all term that includes several different fungal diseases of grapevines trunks worldwide. The term was coined in the late 1990s by Dr Luigi Chiarappa 1, and can include foliar and vascular symptoms caused by Petri disease, Black foot, Eutypa, Botryosphaeria dieback, Phomopsis dieback and Esca. Most of the fungi (more than 50 species) causing trunk disease are related, belonging to the order Botryosphareales, but fungi that belongs to the families that forms mushrooms can be also recovered from the cankers. In most of the cases, the cankers interfere with the movement of water from the roots to the leaves, shots and clusters.
Table 1. Summary of Trunk diseasesPhoto 2. Measles s
Petri disease and Esca are caused by the closely related species of fungi in the genus Phaeomoniella and Paheoacremonium. In both diseases, longitudinal black stripes are visible under the bark. Different from Petri disease which occurs in vines younger than three years, Esca is observed in more mature plants. The foliar symptom can include the well-known “Tiger Stripe” pattern (photo 1). This symptom is visible normally by the end of June, when high temperatures stress the vines. Reddish-brown patches on the leaves are observed in red cultivars, while yellow patches are more common on white grapes. Esca is also known as “black measles” because the dark spots that develop on berries (photo 2). Measles is particularly damaging to table grapes because berry appearance is paramount.
Black foot is caused by two species of the fungal genus Cylindrocarpon, and it most commonly observed on young vines (three-year-old vines or younger). Stunted vines and scorched leaves (photo 3) are a typical sign of black foot. Affected roots present black lesions and look like they are dead. The disease was reported by late 1990’s in California and has been frequently found in fields with poor planting practices, especially those that were J-rooted at planting. The disease if not prevented, can require costly replanting.
Photo 3. Vines affected by black foot disease. Right: Black arrows shows the reduced growth. Left: scorch symptoms.
Botryosphaeria is caused by more than 20 species of fungi. However, fungi in of the species Lasiodiplodia and Neofusicocum are the most damaging. Spurs from infected vines won’t develop new shoots (Photo 4) . In cross-section, a pie shape necrotic lesion can be observed in infected trunks (Photo 5), however this symptom can be also observed in vines infected with Eutypa.
Photo 4. Lack of new shoots growth.Photo 5. Typical symptom of botryosphaeria.
Eutypa is a common disease in California, and it is caused by the fungus Eutypa lata. In addition to the pie shaped internal lesion, proliferation of stunted shoots is common (Photo 6). This symptom is absent in Botryosphaeria disease, where no growth or no leaf symptoms are observed.
Photo 6. Stunted shoots proliferation on vine infected with Eutypa.
Phomopsis is normally associated with damage on green tissue, specially canes and leaves. However, when conditions favor the pathogen, damage caused by Phomopsis can result in dead of spurs, canes and buds. Severely affected canes develop cracks and have a bleached appearance during winter. In early spring reproductive structures of the pathogen are visible as black speckles.
Impact
All the described trunk diseases can be seen at any time of the vineyard life, but normally they start to appear between the third and the fifth year after planting. They can appear alone, or in combination, which can exacerbate the plant stress. In general, by year 10 to 12, 20 percent of the plants show symptoms when no preventive actions have been implemented. At this point, trunk diseases enter in an exponential phase, reaching 75 percent of infection by year 15, and 100 percent by year 20.
Figure 1. Trunk disease infection development in unattended vineyard.
Significant losses are associated with trunk disease development. Reduction in number of clusters, decrease in quality and cosmetic damages are the most visible impacts in the field. However, cost of replanting, and/or retraining if grower elects to use vine surgery, especially in young vineyards, it also a major cost associated with trunk diseases. Siebert2 in 2000, estimated the annual loss caused by Botryosphaeria and Eutypa in the California wine grape industry was $260 million. Similar negative effects have been reported in other grape growing areas and trunk diseases.
Management
Trunk diseases are considered chronic diseases, and unfortunately there is no fungicide that can provide curative action. Preventive management or the removal of infected tissue from the vine (surgery) and destruction by mulching or soil incorporation of infected shoots and canes are the only viable alternatives.
More than 95 percent of trunk diseases infections are associated with pruning, or other cultural practices that leave pruning wounds exposed at a time when the wounds may be infected. Dispersal of the pathogens responsible for causing trunk disease occurs during rain events. Under California conditions pruning and rain overlap during the winter months (November-January). Dr. Gubler and his team found that delaying pruning closer to bud break significantly reduces disease3. The logic behind this is that in a typical California year, rain is gone by the end of February and the days are warmer, letting the plants recover sooner than during the colder days of December and January. In addition, some sap movement (bleeding) starts to be present in February, helping the plant to remove the infective spores from susceptible tissue.
However, and knowing that the labor and logistics doesn’t permit all growers to postpone pruning until the last part of the winter, the use of fungicides to protect the exposed tissues is important to reduce the rate of the infection. The best practice is to protect the plant any time there are pruning wounds. This is especially important if rain is expected following pruning. If pruning is done in November or December, it is advised at least two sprays with protectant fungicides be applied. If the pruning is postponed until January and warmer days are forecasted, one protectant spray after pruning is ideal.
Another strategy for pruning is to do a double pruning (pre-pruning + pruning). It consists of pre-pruning the vines between November and January. Then, by the end of February or March, the pruning process is completed. The objective of this method is to remove any potential infection occurred during the winter months. A complementary fungicide spray after the last pruning can increase the control of trunk diseases.
In order to improve the efficacy of protective fungicide, a closer identification of the disease is recommended as any particular fungicide cannot control all possible pathogens. A recent report done by Baumgartner and Brown4 on their research in 2017, demonstrates that Pristine, Topsin + Rally (in mixture), and Luna had better preventive control of Botryosphaeria and Phomopsis dieback. Eutypa was controlled more effectively with Pristine. The highest control of esca was obtained with Serifel, but it only reached 64 percent. Results in 2018 in the same study presented different results and were mainly associated with a different weather condition.
Different studies, including the one recently done by Dr. Baumgartner and collaborators5, demonstrates that preventive practices works better if they are established during the first years of the crop (Figure 2). Dr. Baumgartner estimated that when more effective practices are adopted early in the crop life, it is expected to prolong the vineyard rentability by at least 25 years.
Figure 2. Calculated lifespan of profitable vineyard based on the efficacy of the implemented practice and the year when they are implemented.
Further information on trunk diseases can be found at:
1. Gramaje D, Úrbez-Torres JR, Sosnowski MR. Managing Grapevine Trunk Diseases With Respect to Etiology and Epidemiology: Current Strategies and Future Prospects. Plant Dis. 2018;102(1):12-39. doi:10.1094/PDIS-04-17-0512-FE
2. Siebert JB. Economic Impact of Eutypa on the California Wine Grape Industry. Davis; 2000.
3. Gubler WD, Rolshausen P e., Trouillase FP, et al. Grapevine trunk Diseases in Califronia. Pract Winer Vineyard. January 2005:1-9.
4. Baumgartner K, Brown AA. Protectants for Trunk-disease management in California table grapes. In: California Table Grape Seminar. Visalia: California Table Grape Commission; 2019:19-21.
5. Baumgartner K, Hillis V, Lubell M, Norton M, Kaplan J. Managing Grapevine Trunk Diseases in California ’ s Southern San Joaquin Valley. 2019;3:267-276. doi:10.5344/ajev.2019.18075
Photo 5: Brown-orange root-like structures allow Rhizopus to rapidly spread between adjacent fruit. All photos courtesy of Steve Koike.
Growers face a multitude of obstacles when trying to produce large volumes of high-quality strawberry fruit for a market that runs for many months. Up-front, pre-plant ground preparation and transplant costs are significant financial commitments that are expensed before even a single strawberry plant is put in the ground. Untimely rains and insect infestations can result in loss of fruit quality and numbers. A series of soilborne pathogens can later cause plant collapse and loss of profit. Of course, the intractable labor shortage dilemma may even result in perfectly marketable fruit not reaching the consumer.
Photo 1: Two pathogens, Rhizopus and Mucor, can turn strawberry fruit into black lumps.
When it comes to diseases of the strawberry fruit, the number one concern is gray mold (also called Botrytis fruit rot), which justifiably attracts the rapt attention of field professionals and captures the interest of researchers. However, in coastal California another fungal issue can also take its toll on strawberry yields and quality and in many ways is overlooked and underestimated by the industry. Rhizopus fruit rot and Mucor fruit rot are collectively known as “leak” disease; this disease concern also deserves to be recognized and studied.
Symptoms, Signs, Diagnosis
In the field, leak symptoms and signs only develop on mature or near-mature fruit and are very distinctive. Infected red fruit first take on a darkened, water-soaked appearance; in short order the fruit will begin to wrinkle and collapse. Almost overnight the rapidly growing Rhizopus or Mucor pathogen will be visible as white fungal growth peppered and interspersed with tiny black spheres. Fungal growth will be extensive and can entirely envelop the fruit, turning it into a white and black lump (Photo 1). Rhizopus and Mucor produce pectolytic (endopolygalacturonase) and cellulase enzymes as they colonize the strawberry, disintegrating the fruit tissues and causing red juices (Photo 2) to ooze and flow onto the plastic that covers the bed. It is because of these messy red juice flows that the designation “leak” is used for this disease.
Table: Description of various fungi found on post-harvest strawberry.
This ugly scene in the field, however, is only part of the problem that these fungi cause. During harvest infected but symptomless fruit, as well as healthy fruit exposed to spores of the two pathogens, will be packed into containers. The pathogens can continue to grow during postharvest handling, storage, and market display of fruit, causing postharvest fruit losses, shortening of shelf life of the product, and creating a mess in crates and clamshells (Photo 3). In fact, if fruit are not properly refrigerated, the fungus on a single infected fruit can rapidly spread throughout an entire container, resulting in what is known as “nesting” or clumping of oozing, rotted fruit. Harvested strawberry fruit are subject to a number of rotting molds; the leak fungi are generally identifiable due to the color and nature of the fungal growth (see table).
The Pathogens
Much is already known about the two fungi causing strawberry leak disease. Rhizopus and Mucor are both in the group of fungi called Zygomycetes. Both fungi are commonly found in agricultural environments and cause similar ripe fruit rots on crops such as apricot, cherry, peach, pear, and tomato. While closely related to each other and difficult to differentiate in the field, the two pathogens differ slightly. On California strawberry, Rhizopus tends to be the more commonly encountered pathogen. Rhizopus grows very rapidly and haphazardly in orientation, creating a web-like mess of mycelium (Photo 4). Rhizopus forms a brown orange, root-like structure (called rhizoids) that allows it to quickly spread between adjacent fruit (Photo 5). The black, spherical spore bearing structures produce huge numbers of dry spores that are readily spread by winds (Photo 6). Mucor grows more slowly with an upright, erect mycelial habit (Photo 7) and also produces spores on a black spherical structure (Photo 8). However, Mucor spores collect in a wet droplet surrounding the head; such spores are therefore less prone to dispersal by winds.
Photo 2: The leak pathogens produce enzymes that cause strawberry fruit to ooze juice.Photo 3: Enzymes released by Rhizopus and Mucor can cause significant fruit breakdown during storage and transport.
Both fungi survive and increase in the field by colonizing dead organic matter and debris; Rhizopus and Mucor also readily colonize discarded and over-ripe strawberry fruit left on the plant or thrown into the furrow. These fungi produce a resilient structure (zygospore) that resists drying and weathering and provides another means of survival. Both Rhizopus and Mucor can be readily isolated from soil, thereby demonstrating that these fungi can persist in fields even if strawberry is not present. Once strawberry fruit begin to develop and ripen, spores come in contact with the fruit and usually gain entry via wounds and injuries. Strawberry leak pathogens tend to be more active if temperatures are relatively warmer, generally 65 F or higher. This temperature factor may be one reason that leak disease is more damaging in coastal California in late summer through early fall. The pectolytic enzyme that causes fresh market fruit to melt into juices can also be a concern in some processed fruit products. The enzyme is heat-stable and withstands canning temperatures; for example, apricot halves and brined cherries that are contaminated with Rhizopus and then canned may end up being apricot or cherry mush because of the continued activity of the stable enzyme even though the original fungus is cooked and dead.
Photo 4: Rhizopus growth is rapid and results in a messy, web-like mycelial growth.
Management Options and Research Needs
Growers already know to refrigerate strawberries as soon as possible after field packing the fruit. Refrigeration is a primary management tool for reducing losses due to leak disease. While refrigeration generally limits the development of Rhizopus, it is notable that some species of Mucor can grow quite well at storage temperatures of 32 F (0 C). Therefore, one research need is the precise identification of Rhizopus and Mucor species present in strawberry fields. Different species will have different temperature optima regarding growth and ability to infect fruit in the field, and the different species may respond differently to postharvest conditions.
Photo 6: The black, spherical spore-producing head of Rhizopus releases thousands of wind-borne spores.
Sanitation, both in field and during harvest, is another means of limiting leak development. Reducing the amount of over-ripe and rotted fruit in the field may help limit the Rhizopus and Mucor inoculum present in the planting; the influence of field sanitation on inoculum is another area of needed research. Field sanitation may be an increasingly difficult goal to attain given labor shortages and costs. Sanitation during the harvest process mainly involves the training and education of harvesters. Harvesters who touch and handle leak fruit will easily transmit the fungus to healthy fruit that are packed. Therefore, pickers should be reminded not to touch leak fruit and to be sure not to pack fruit showing any hint of leak infection.
Photo 7: On strawberry fruit, the Mucor pathogen grows with an upright, erect mycelial habit.
Fungicides can effectively limit Rhizopus development for some crops. However, such information is lacking for strawberry. Research is needed to determine the efficacy and feasibility of using fungicides for leak management in strawberry. Other interactions involving fungicides should also be investigated. There are indications that fungicides used to manage Botrytis could exacerbate growth by Rhizopus and Mucor.
Photo 8: Mucor also produces a black, spherical head, though the spores are captured in a droplet and are not readily spread by wind.
Among the many challenging factors facing strawberry growers, leak disease is not the number one concern. However, during certain times of the year in coastal California, leak disease can affect a significant amount of the harvest. A better understanding of strawberry leak disease, achieved through collaborative research, may help this industry manage this problem and improve on an already excellent commodity.
Strawberries are the third most valued crop in California ($2.3 billion) and one of the most sensitive to salinity. Limited information on the tolerance of new varieties to salt and chloride toxicity has led to significant yield losses in recent years. Even a modest yield loss of 5 percent due to soil and water salinity may cost the strawberry industry and California $260 million per year. Despite the importance, commonly used salinity tolerance thresholds for strawberry (Ayers and Westcot, 1985) are based on studies almost a half-century old and may not be applicable to the soils, water quality, climate, and modern cultivars grown in California. California production, which accounts for approximately 85 percent of the strawberries produced in the US, are mostly grown on coastal soils with electrical conductivity (ECe, saturated paste extract method) ranging from 2 to 4 dS/m. Some of these soils have moderate to high concentrations of calcium, bicarbonate and sulfate. Although most of these salts may be precipitated in the form of calcium sulfate (gypsum) and calcium carbonate (lime) and have limited impact on plant growth, the ECe can be considerably increased when a soil sample is saturated with distilled water (used in the saturated paste extract method) due to the dissolution of these salts. That is a common scenario found in arid regions such as in the Southwestern US (e.g. Ventura County), where limited rainfall contributes to the accumulation of certain salts in the topsoil. In the Watsonville area, however, there is much less carbonate and sulfate in the groundwater and therefore in agricultural fields, where sodium and chloride are of major concern. Excessive sodium most often leads to high sodium adsorption ration (SAR), which causes infiltration problems on some soil types. In irrigated agriculture, the irrigation water quality and leaching fraction are usually the main factors driving soil salinity. When appropriate leaching amounts are applied, the salinity of the soil and of the irrigation water reach a steady-state (equilibrium). However, when irrigation amounts do not exceed crop evapotranspiration (ETc), soil salinity can increase considerably due to the accumulation of salts in the rootzone. Infiltration rate and rainfall also affect soil salinity. In an attempt to account for the precipitation effect of certain salts, Rhoades et al. (1992) suggest that plants can tolerate ECe about 2 dS/m higher than published thresholds when grown on gypsiferous soils (soils that contain significant quantities of gypsum, or calcium sulfate). Although this publication provides basic management guidance, strawberry growers and farm managers need more detailed information to determine leaching fractions and to select irrigation water sources and cultivars. Identifying the specific types of anions and cations that make up the salts in soil and irrigation water is important for predicting how different strawberry cultivars will tolerate salinity. For example, a field with soil ECe of 2.5 dS/m, where chloride is approximately 10 meq/L can have significantly greater impacts on strawberry yields than a soil with the same soil ECe where chloride is 2 meq/L and calcium and sulfates are the predominate salts.
Table 1. Irrigation water chemical analysis average and standard deviation of 40 fields in the Oxnard and in the Watsonville production districts, year 1 (2016/2017 season).
Material and Methods
In order to assess how susceptible strawberry cultivars are to irrigation water of different quality, a two-year study was conducted in California between 2016 and 2018. The first year of the study consisted of a survey conducted in 40 strawberry fields located in the Oxnard and Watsonville districts, where irrigation water and soil samples were analyzed for salinity composition. Overall, the irrigation water of the fields located in the Oxnard district had greater electrical conductivity and significantly greater sulfate levels, while chloride levels in the Watsonville fields were twice as great as the Oxnard fields (Table 1). These results were used as the benchmark for determining the treatments of the salinity tolerance experiment conducted the following year.
Figure 1. Picture of experiment site with water-powered injectors mounted on top of 55 gallons containers of each treatment, with experiment plots in the background.
The second year of the study consisted of an experiment conducted in a commercial field located in Oxnard, California during the 2017/2018 production season. Strawberry yield, soil salinity and salts content in leaf blades of the two most popular public cultivars in Oxnard (cv. Fronteras) and in Watsonville (cv. Monterey) were assessed under eight salinity treatments following a randomized complete block design. Each plot was 30 feet long and 1 bed wide (64 inches), with four plant rows, approximately 90 plants/plot, and two high flow drip tapes (0.67 gpm/100ft) placed about 1.5 inch deep between the 1st and 2nd, and between 3rd and 4th plant rows. The experiment was planted on October 2017, and the treatments started approximately a month post-planting in order to promote a good establishment of the crop; during that period, overhead micro-sprinklers was the predominant irrigation method. Drip irrigation amounts and timing were decided based on ETc estimations from the California Irrigation Management Information System (CMIS station # 152) weather station, and matric potential readings from tensiometers, respectively. Water-powered injection pumps (Figure 1) blended the well water with concentrated salt solutions formulated for each treatment at a 1:100 ratio during every drip irrigation event from November 2017 to June 2018 (total of 60 drip irrigations). The treatments consisted of irrigation water with two levels of elevated sodium adsorption ratio (SAR, 4.6 and 6.6), three levels of elevated chloride (4.2, 7.7 and 11.7 meq/L), and two levels of elevated sulfate (18.3 and 26 meq/L of SO4). Table 2 displays a complete description of the salts’ composition of each treatment. Composite soil and leaf blade samples were collected from each plot at early, mid and late production stages and analyzed for pH, ECe, Ca, Mg, Na, Cl, B, HCO3, CO3 and SO4 (soil samples), and N, P, K, S, B, Ca, Mg, Zn, Mn, Fe, Cu, Na and Cl (leaf blade samples). Marketable and unmarketable yield, and berry weight were measured in average twice a week from December 2017 through June 2018, totaling 54 harvesting events. There was a total of 5.8 inches of rainfall throughout the entire growing season, of which 4.8 inches happened in March, between the first and second sampling events.
Table 2. Chemical analysis of irrigation water treatments of year 2 (2017/2018 season). Values represent average of three samples collected from the drip tape throughout the season.
Results and Discussion
Total marketable yield of Fronteras was significantly (P<0.05) reduced by 13 and 17 percent with increasing chloride levels of 7.7 and 11.7 meq/L, respectively (Table 3). Although yields of all other treatments were lower than the control treatment (Figure 2), those differences were not statistically significant (P>0.05). The high sulfate treatment reduced Fronteras cv. marketable yield by 10 percent, although the differences were not statistically significant (P=0.094). Yield losses due to the elevated salts started before plant symptoms were noticeable in Fronteras. Total marketable yield of the Monterey cultivar was not significantly affected by any salinity treatment (Figure 3 and Table 4). Cull rates of both cultivars were not affected by the salinity treatments. Fronteras berry weight was significantly reduced by 6.3 percent with the highest chloride treatment (11.7 meq/L). Salt concentrations in soil and leaf blade samples consistently increased with the higher salinity treatments for both cultivars (data not shown).
Table 3. Total yield response to treatments, Fronteras cultivar.Figure 2. Total marketable yield of Fronteras cultivar displayed in boxplot graph; in this graph, the box represents the limits between the 25th and the 75th percentiles, and the whiskers represent the upper and lower endpoints. The horizontal line inside the box represents the median.
The rainfall events that occurred between the first and second samplings contributed to significant leaching of salts from the root zone (0-12 inch depth), which made overall ECe values from the second sampling date very similar to the values measured during the first date. Hence, yield losses observed in this experiment may have been greater, and occurred sooner if the rainfall during that period had been less. Additionally, plant symptoms of the salinity treatments, which were not observed until mid-May, may have been observable sooner with less intense precipitation. Overall, the most surprising finding of this study is the marked differences in yield response to the salinity treatments between the two cultivars. While Fronteras proved to be highly susceptible to increased irrigation water salinity, especially in regards with chloride, Monterey cv. presented limited and statistically not significant yield declines. Accordingly, other major public and proprietary strawberry cultivars may also exhibit a range of susceptibility to salinity. It is also reasonable to expect greater yield losses of the cultivar Fronteras grown on fields that have been farmed with irrigation water quality equivalent to the treatments of this study for years. In that case, plant establishment can be compromised by the increased water and soil salinity, especially if the irrigation wasn’t managed with the appropriate leaching requirement to the water quality. The fact that the Monterey cultivar did not respond to the salinity treatments included in this study may be related to significantly greater chloride levels found in the irrigation water of the Watsonville area, where the cultivar was selected and tested before being released to commercial production.In summary, the findings of this study conclude that the strawberry cultivar Fronteras is highly susceptible to elevated chloride levels, and that salinity effects on strawberry yield is cultivar dependent. Although this study provides conclusive information of salinity effects on Fronteras and some information about Monterey cultivar, the quest to understanding the impact of salts on the main strawberry cultivars is very challenging and most likely far from being achieved.
Figure 3. Total marketable yield of Monterey cultivar displayed in boxplot graph.Table 4. Total yield response to treatments, Monterey cultivar.
References
Ayers, R.S. and D.W. Westcot, 1985. FAO Irrigation and Drainage Paper 29: Water quality for agriculture. In Crop tolerance to salinity.http://www.fao.org/3/T0234E/T0234E03.htm#ch2.4.3
Rhoades, J.D., A. Kandiah and A.M. Mashali. 1992. FAO Irrigation and Drainage Paper 48: The use of saline waters for crop production:http://www.fao.org/3/t0667e/t0667e00.htm
