Antarctic fungi found to be effective against citrus canker
Brazilian researchers have identified activity against Xanthomonas citri in 29 fungi isolated from samples collected in Antarctica; one of the compounds inhibited reproduction of the bacterium by up to 98 percent
A research team at the São Paulo State University's Bioscience Institute (IB-UNESP) in Rio Claro, Brazil, has identified 29 fungi with proven action against Xanthomonas citri, a bacterium responsible for citrus canker, an endemic disease in all citrus-producing countries. The origin of the fungi is surprising. They were isolated from samples of soil and marine sediment collected in Antarctica.
"These fungi live in isolated conditions and proliferate under inhospitable conditions including low temperatures and high levels of ultraviolet radiation," says Daiane Cristina Sass, a Professor at UNESP who heads a project engaged in a search for microorganisms that produce compounds with antibacterial action for use in agriculture, with support from the São Paulo Research Foundation - FAPESP.
"How have they adapted to survive in an environment so hostile to life? We wanted to see if they produced molecules with unique structures that protected them from infections and might therefore be capable of antibacterial action." Sass wrote an article published in Letters in Applied Microbiology - jointly with IB-UNESP colleagues Lara Durães Sette and Henrique Ferreira, among others - which shows some of the research's results.
More efficiency on fighting citrus canker
Although the bacterium can be combated in several ways, none is sufficient to eradicate the disease. Therefore, new chemical or biological methods of protecting citrus groves have to be pursued.
The disease is controlled directly by growers. The recommended measures include spraying trees with copper-based products and replacing infected trees with healthy new plantings derived from more resistant varieties. Control of the citrus leaf miner (Phyllocnistis citrella) is also advisable. The wounds made by larvae of this moth in feeding on the plant exacerbate citrus canker by serving as an entry point for X. citri.
"The main method for combating citrus canker consists of spraying trees with copper compounds. The downside is that when even small amounts are used for a long period, copper accumulates in the fruit, soil and water, eventually contaminating the entire environment. For this reason, we're looking for new compounds that are less aggressive to the environment and also less harmful to humans," Sass explained.
Collection and isolation of the Antarctic fungi
On the extent of the Sass-headed project and its research on biotechnology, the team came up with the idea of investigating the collection of fungi curated by Professor Sette, which resulted from Antarctic summer expeditions to the South Shetland Islands in 2013 and 2015 as part of Project Microsfera, conducted under the aegis of the Brazilian Antarctic Program (PROANTAR) and sponsored by the National Council for Scientific & Technological Development (CNPq).
Sette leads the project "Marine and Antarctic mycology: diversity and environmental application", also supported by FAPESP.
Sette isolated 33 filamentous fungi from samples collected in soil under rotten wood on Deception Island and 53 filamentous fungi from marine sediments at a depth of 20 meters in Admiralty Bay, King George Island. All fungal strains are kept at UNESP's Microbial Resource Center (CRM).
The FAPESP-funded research found that 29 of the 86 Antarctic fungi they isolated (19 of marine origin and ten terrestrial) contained compounds with proven action against X. citri.
Isolating the compounds produced by the fungi and verifying their antibacterial activity involved several stages. The process began with isolation of the fungi, which were then grown for several days in culture dishes with nutrients.
The fungi were cultured in liquid medium and shaken for 20 days at 15 °C. The solid biomass was separated from the liquid portion, and both parts were submitted to processing with solvents to obtain intracellular and extracellular extracts.
The researchers obtained 158 extracts. Each extract was diluted at several concentrations (2.10 mg/ml-0.02 mg/ml) and tested against X. citri. In the case of the soil fungi, most of the extracts with antibacterial action were intracellular in origin, while for the marine fungi, only the extracellular extracts hindered the bacterium's growth.
"We wanted to determine the lowest concentration of each extract that inhibited growth in 90% of cases," Sass said.
Some (12) of the extracts affected bacterial growth at lower concentrations than the highest tested, and ten of these inhibited growth in more than 90% of cases at concentrations of 1.5 mg/ml-1.0 mg/ml.
"At maximum concentration, one extract inhibited growth by up to 98%, and another inhibited it by about 80% at 0.52 mg/ml," Sass said. "It's important to note that we're talking about extracts [which contain varying amounts of molecules]. If an extract contains only one compound that's responsible for this bioactivity, the compound may display good antibacterial activity at much lower concentrations."
Twenty of the isolated fungi with action against X. citri belonged to the genus Pseudogymnoascus and were extracted from terrestrial and marine samples. Next came Penicillium (five), followed by Cadophora (two), Paraconiothyrium (one) and Toxicocladosporium (one), all extracted from marine sediments.
Having identified the extracts with action against X. citri, the researchers are now working to find out which chemical compounds give them this antibacterial capability.
"We expect to identify and purify some of these bioactive compounds, as well as to complete toxicology testing on them, within 18 months or less," Sass said.
The researchers plan to patent the compounds they identify. They also hope to persuade pesticide manufacturers to develop commercial products for combating citrus canker based on these compounds.
Twenty of the isolated fungi with action against X. citri belonged to the genus Pseudogymnoascus and were extracted from terrestrial and marine samples. Next came Penicillium (five), followed by Cadophora (two), Paraconiothyrium (one) and Toxicocladosporium (one), all extracted from marine sediments./h1>
Drought Induced Problems in Our Orchards
Abiotic disorders are plant problems that are non-infective. They are not caused by an organism, but through their damage, they may bring on damage caused by organisms. Think of a tree hit by lightning or a tractor. The damage breaches the protective bark which allows fungi to start working on the damaged area, eventually leading to a decayed trunk. It was the mechanical damage, though that set the process in motion.
Too much or too little water can also predispose a plant to disease. Think of Phytophthora root rot or even asphyxiation that can come from waterlogging or too frequent irrigations.
Salinity Effects from Lack of Water
Lack of water and especially sufficient rainfall can lead to salinity and specific salts like boron, sodium and chloride accumulating in the root zone. This happens from a lack of leaching that removes native soil salts from the root zone or the salts from the previous salt-laden irrigation from the root zone. These salts cause their own kind of damage, but they can also predispose a tree to disorders, disease and invertebrate (insect and mite) damage.
Lack of water and salt accumulation act in a similar fashion. Soil salt acts in competition with roots for water. The more soil salt, the harder a tree needs to pull on water to get what it needs. The first symptom of lack of water or salt accumulation may be an initial dropping of the leaves. If this condition is more persistent, though we start to see what is called “tip burn” or “salt damage”. Southern California is tremendously dependent on rainfall to clean up irrigation salts, and when rain is lacking, irrigation must be relied on to do the leaching
As the lack of leaching advances (lack of rainfall and sufficient irrigation leaching) the canopy thins from leaf drop, exposing fruit to sunburn and fruit shriveling.
Leaf drop and fruit shriveling in avocado.
In the case of sensitive citrus varieties like mandarins, water stress can lead to a pithy core with darker colored seeds, almost as if the fruit had matured too long on the tree.
Total salinity plays an important factor in plant disorder, but also the specific salts. These salts accumulate in the older leaves, and cause characteristic symptoms that are characteristic in most trees. Boron will appear on older leaves, causing an initial terminal yellowing in the leaf that gradually turns to a tip burn.
Often times it is hard to distinguish between chloride, sodium and total salinity damage. It is somewhat a moot point, since the method to control all of them is the same – increased leaching. There is no amendment or fertilizer that can be applied that will correct this problem. The damage symptoms do not go away until the leaf drops and a new one replaces it. By that time hopefully rain and/or a more efficient irrigation program has been put in place.
The Impact of Drought on Nutrient Deficiencies
Salinity and drought stress can also lead to mineral deficiencies. This is either due to the lack of water movement carrying nutrients or to direct completion for nutrients. A common deficiency for drought stressed plants is nitrogen deficiency from lack of water entraining that nutrient into the plant.
This usually starts out in the older tissue and gradually spreads to the younger tissue in more advanced cases.
The salts in the root zone can also lead to competition for uptake of other nutrients like calcium and potassium. Apples and tomatoes are famous for blossom end rot when calcium uptake is low, but we have also seen it in citrus. Low calcium in avocado, and many other fruits, leads to lower shelf life. Sodium and boron accumulation in the root zone can lead to induced calcium deficiencies and increased sodium can also further lead to potassium deficiencies. Leaching can help remove these competitive elements.
Drought Effect on Tree Disease
Drought and salt stress can also lead to disease, but in many cases once the problem has been dealt with the disease symptoms slowly disappear. They are secondary pathogens and unless it is a young tree (under three years of age) or one blighted with a more aggressive disease, the disease condition is not fatal. Often times, in the best of years, on hilly ground these diseases might be seen where water pressure is lowest or there are broken or clogged emitters. The symptoms are many – leaf blights, cankers, dieback, gummosis – but they are all caused by decomposing fungi that are found in the decaying material found in orchards, especially in the naturally occurring avocado mulch or artificially mulched orchards. Many of these fungi are related Botryosphaerias, but we once lumped then all under the fungus Dothiorella. These decay fungi will go to all manner of plant species, from citrus to roses to Brazilian pepper.
Another secondary pathogen that clears up as soon as the stress is relieved is bacterial canker in avocado. These ugly cankers form white crusted circles that ooze sap, but when the tree is healthy again, the cankers dry up with a little bark flap where the canker had been.
Drought Effect on Pests
Water/salt stress also makes trees more susceptible to insect and mite attack. Mites are often predated by predacious mites, and when there are dusty situations, they can't do their jobs efficiently and mites can get out of hand. Mite damage on leaves is often noted in well irrigated orchards along dusty picking rows
Many borers are attracted to water stressed trees and it is possible that the Polyphagous and Kuroshio Shot Hole Borers are more attracted to those trees.
And then we have conditions like Valencia rind stain that also appears in other citrus varieties. We know it will show up in water stressed trees, but we aren't sure what the mechanism that causes this rind breakdown just at color break. Could it be from thrips attracted to the stressed tree or a nutrient imbalance, it's not clear?
Water and salt stress can have all manner of effects on tree growth. It should lead to smaller trees, smaller crops and smaller fruit. The only way to manage this condition is through irrigation management. Using all the tools available, such as CIMIS, soil probes, soil sensors, your eyes, etc. and good quality available water are the way to improve management of the orchard to avoid these problems.
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Tip Burn, notice sun burn bottom right hand fruit
Endoxerosis with dried out core
Blossom end rot
Bot gumming in lemon
Black Streak in Avocado
Citrus red mite
Polyphagous Shot Hole Borer damage on avocado
Valencia Rind Stain
There have been so many calls recently with the same problem, thinning canopy with dieback. Thiscan be caused by several problems, but the most common this time of year is lack of good water management over the year, accentuated by lack of rain and salt damage. The rain will often leach salts accumulated from the root zone over these many years of water from only irrigation. I thought as everyone else did that we would be having some gushers in S. California and some of these problems would be relieved. With rain, the cankers from black streak, bacterial canker and Botryosphaeria canker (Dothiorella) staining are washed away, and often times, so are the tree's problems. Well that hasn't happened. Or rather the rain hasn't happened to resolve this problem - Botryosphaeria or Bot Rot.
This problem has been ongoing and growers are still calling in about avocados with thinning canopies, fruit drop and sunburn and leaf death. Coastal avocados are always difficult to irrigate. Mild weather followed by dry windy conditions means growers have to scramble to get water on. If the trees are on some sort of calendar schedule, it usually means the trees get stressed. If trees are on a slope where pressure is not properly regulated, some of the trees are going to get stressed. If emitter clogging is not addressed, then more trees get stressed. And lack of rains to leach salts from the root zone, and more trees are stressed.
This stress sets up the trees for disease and a very common one in an avocado orchard that is filled with lots of leaves where decay fungi are working is stem and leaf blight. The disease causes defoliation and exposed fruit sun burn and drop. In an orchard, it's possible to see healthy trees and sick ones at the same time. This may be due to the differences in soil type from tree to tree or the fruit load on trees - more fruit, more stress. Looking out over the orchard there may be a polka dot of sick trees. And it might all happen at once in a week or gradually.
So, this is a problem that is out there, if the irrigation issue is corrected, the trees usually recover. It might require white washing and pruning out dead tissue. If it is a young tree under two years, it might actually kill the little tree, but the disease is not usually fatal, just loosing the fruit.
This is a disease that goes to many different tree species - redwoods, eucalyptus, pine, Brazilian pepper, CITRUS. The cause is the same, water or salinity stress. To read more about this disease, go to:
Thinning canopy, dieback and shriveled fruit in avocado and dieback and gumming in citrus
GAINESVILLE, Fla. — Researchers from the Institute of Food and Agricultural Sciences at the University of Florida are closer to finding a possible cure for citrus canker after identifying a gene that makes citrus trees susceptible to the bacterial pathogen.
Citrus canker, which causes pustules on fruit, leaves and twigs, is a highly contagious plant disease and spreads rapidly over short distances. Wind-driven rain, overhead irrigation, flooding and human movement can spread citrus canker. Human transport of infected plants or fruit spreads the canker pathogen over longer distances.
In Florida, the last extensive canker outbreak occurred beginning in 1995, which led to an ultimately unsuccessful eradication program that ended in 2006. That effort cost an estimated $1 billion and stimulated renewed efforts for more effective and economical controls. Farmers destroyed more than 16.5 million citrus trees between 1995 and 2012.
Yang Hu, a former doctoral student working with Jeff Jones, a professor in plant pathology, found the critical trait in the bacterium that is necessary for disease development. Hongge Jia, a researcher at UF's Citrus Research and Education Center in Lake Alfred, and Nian Wang, an associate professor in microbiology and cell science also based at the Citrus REC, along with six researchers from three universities worked on the project, as well.
Citrus canker continues to be a problem and exists in most citrus-growing areas in Florida. While scientists like Hu are devoted to eradicating the disease, many other researchers are now also battling citrus greening, which threatens to wipe out the $9 billion industry.
Citrus canker is caused by the bacteria Xanthomonas citri. While studying the bacterial pathogen's role in infected citrus, researchers were able to identify a gene in citrus critical to the development of citrus canker, known as the susceptibility, or “S” gene.
By finding the susceptibility gene, researchers say they are closer to a cure for the disease.
“The S gene represents an excellent candidate for control measures for the citrus bacterial canker,” Hu said.
Hu and Jones said they hope to secure funding to support further research, and have already identified several genes they believe could be engineered to obtain broad-spectrum plant resistance to most kinds of citrus canker.
“Once you know what the susceptibility gene is, it's possible to design multiple strategies for disease control,” Jones said.
The research paper was published online this month by Proceedings of the National Academy of Sciences: http://www.pnas.org/content/early/2014/01/08/1313271111.abstract.
- Author: Akif Eskalen and Virginia McDonald
Branch and trunk canker on avocado was formerly attributed to Dothiorella gregaria, hence the name Dothiorella canker. So far Botryopshaeria dothidea (anamorph: Fusicoccum aesculi) is the only known species causing Dothiorella canker on avocado in California. Symptoms observed on avocado with Dothiorella canker include shoot blight and dieback, leaf scorch, fruit rot, and cankers on branches and bark.
However, recent studies based on DNA analyses suggest greater species diversity of this pathogen group than based on morphological characteristics alone. Thus far, multiple species of Botryosphaeriaceae have been found to cause the typical Dothiorella canker (Fig3.) and stem-end rot (Fig 5) on avocado in California. Percent recovery of Botryosphaeria spp. based on morphological characters ranged from 40-100% in Riverside county, 42-53% in Ventura county, 33% in Santa Barbara county, 60% in San Diego county and 32-60% in San Luis Obispo county.
According to preliminary results from a continuing survey throughout avocado growing areas of California, multiple species of Botryosphaeria (Neofusicoccum australe, B. dothidea, N. luteum, and N. parvum) were found.
Pycnidia (overwintering structure) of Botryosphaeriaceae species were also observed on old diseased avocado tree branches. Sequenced rDNA fragments (ITS1, 5.8S rDNA, ITS2, amplified with ITS4 and ITS5 primers) were compared with sequences deposited in GenBank.
Pathogenicity tests were conducted in the greenhouse on 1-year-old avocado seedlings, Hass cv., with one randomly chosen isolate from each of the Botryosphaeriaceae species noted above. Four replicate seedlings were stem-wound inoculated with a mycelial plug and covered with Parafilm. Sterile PDA plugs were applied to four seedlings as a control. Over a period of 6 months, seedlings were assessed for disease symptoms that included browning of leaf edges and shoot dieback. Mean vascular lesion lengths on stems were 64, 66, 64, and 18 mm for B. dothidea, N. parvum, N. luteum, and N. australe, respectively. Each fungal isolate was consistently reisolated from inoculated seedlings, thus completing the pathogenicity test. To our knowledge, this is the first report of N. australe, N. luteum, and N. parvum recovered from branch cankers on avocado in California.
These results are significant because Botryosphaeriaceae canker pathogens are known to enter the host plant through fresh wounds (pruning, frost, and mechanical). With high-density planting becoming more common, which requires intensive pruning, the transmission rate of these pathogens could increase in California avocado groves. The Eskalen laboratory is currently investigating control measures for dothiorella canker and stem-end rot pathogens.
Branch dieback and trunk canker caused by the fungus