Home
Calendar
Search
Contact Us
Webmaster
 

Cultural Control and Etiology of Replant Disease of Prunus spp.

Final Report, 2002

 


Principal Investigator (main contact):
Greg Browne
USDA-ARS, Department of Plant Pathology
One Shields Ave., University of California, Davis
Davis, CA 95616-8680,
Email: gtbrowne@ucdavis.edu

Other Investigators:
Tom Trout
USDA-ARS, Water Management Research Laboratory
9611 S. Riverbend Ave.
Parlier, CA
Email: ttrout@fresno.ars.usda.gov

Russ Bulluck
APHIS, Raleigh, North Carolina
c/o Department of Plant Pathology
One Shields Ave., University of California, Davis
Davis, CA 95616-8680


Cooperators:
Joseph Connell
Farm Advisor
UCCE Butte County
2279-B Del Oro Ave
Oroville, 95965
Email: jhconnell@ucdavis.edu

Sally Schneider
USDA-ARS, Water Management Research Laboratory
9611 S. Riverbend Ave. Parlier, CA
Email: sschneider@fresno.ars.usda.gov

Steve McLaughlin
Department of Plant Pathology
One Shields Ave.University of California, Davis
Davis, CA 95616-8680
Email: stmclaughlin@ucdavis.edu

Harold Becherer
USDA-ARS, Department of Plant Pathology
One Shields Ave., University of California, Davis
Davis, CA 95616-8680
Email: hebecherer@ucdavis.edu

Andreas Westphal
Botany & Plant Pathology Department
Purdue University
1155 Lilly Hall of Life Sciences
West Lafayette, IN 47907-1155
Email: westphal@purdue.edu

Garry Pearson
Department of Vegetable Crops
One Shields Ave., University of California, Davis
Davis, CA
Email: pearson@vegmail.ucdavis.edu

Jim Paiva
Paiva Farms
13522 Hamilton Nord Cana Highway
Chico, CA 95973,
Phone: 530-345-8491

Ed Hosoda
Cardinal Professional Products
10 N. East St.
Woodland, CA, 95776-5922
Email: ehosoda@pacbell.net


Husein Ajwa
Dept. of Vegetable Crops
UC Davis, c/o USDA-ARS
1636 East Alisal St.
Salinas, CA 93905
Email: haajwa@ucdavis.edu


Project locations:
Lab and greenhouse work at University of California, Davis, Yolo County
Current Field trials near Chico, Butte County; and Parlier, Fresno County


Commodities:
Prunus species, including almond, peach, and plum


Funding:
Year 1: $51,948 (SAREP)
Year 2: $49,993 (SAREP), $6,000 (Fruit Tree, Nut Tree, and Grapevine Improvement Advisory Board), and $10,000 (Almond Board of California)
Year 3: $48,697 (SAREP) and $10,000 (Almond Board of California)

Table of Contents:

Objectives
Summary
Specific Results
Potential Benefits/Impacts on Agriculture
Dissemination of Findings
Tables
Figures

Objectives

The overall goal of the research has been to reduce dependence on pre-plant fumigation with methyl bromide (MB) for control of replant disease (RD). The specific objectives were to:
1. Determine effects of pre-plant bare-fallow periods and pre-plant cover cropping on development of replant disease (RD) on peach in California.
2. Determine if cross-specificity exists between RD of peach and grape.
3. Determine organisms and factors that cause RD on selected Prunus spp. in California.

Summary

Replant disease (RD, also known as replant disorder) of Prunus species can complicate establishment of stone fruit and nut orchards planted after removal of a closely related crop. It results in poor growth, delayed crop production, and, in severe cases, tree death (Fig 1). RD is most clearly evident when it occurs in the absence of known causes of other replant problems, which can include plant-parasitic nematodes, Armillaria mellea, Phytophthora species, Verticillium dahliae, or chemical or physical soil inadequacies. It can be prevented by pre-plant fumigation with methyl bromide (MB). Past research indicates that RD, as it is referred to here, can severely limit tree performance even in the absence of the known causes for replant problems. Current data indicate that the cause(s) for RD are primarily biological; RD symptoms can be prevented by soil heating (50 to 60 C) or by treating soil with diverse fumigants. The research on Prunus RD reported here was conducted at sites or with soils that lacked significant populations of plant parasitic nematodes.

Effects of 0- to 3-year pre-plant fallow periods on performance of peach and plum trees on old orchard sites were determined in four experiments conducted by the USDA-ARS Water Management Research Lab near Parlier, CA. In each test, a pre-plant MB fumigation treatment (350 lb/A, 0-year fallow) was included as a standard, and trunk growth and marketable fruit yields were used to assess treatment benefits. Without fumigation, each additional year of pre-plant fallow from 0 to 3 year incrementally increased the amount of tree growth produced during the first several years after planting. However, not all fallow-induced growth increases were statistically significant, and they were not all accompanied by significant increases in first year marketable fruit yields. The results indicated that at least 3 year of pre-plant fallow are needed to match the growth and first-harvest yields produced following pre-plant fumigation with MB. It was not clear that 1 year of fallow provided a significant yield benefit, compared to no fallow, but growers should consider that optimal cultural preparation for replanting often requires a year of fallow.

Effects of pre-plant cover cropping on RD were investigated in two greenhouse experiments and are now being tested in field micro plots. For the greenhouse tests, soil samples from old peach and plum sites were planted with 10 different cover crops in pots in a greenhouse. Non-cropped and MB treatments were included for comparison. After 4 mo of growth, the cover crops were shredded, incorporated into the soil, and allowed to decompose for 1 month. Nemaguard peach seedlings were transplanted in all of the soils and used to assay them for RD potential. In these tests there was no consistent effect of pre-plant cover cropping or fumigation on peach plant performance or incidence of root-associated fungi on peach. It is not certain that the greenhouse tests adequately represented field settings. To address the potential shortcomings of greenhouse tests, field micro plots were established in 2002. The micro plots, filled with soil from a peach RD site, received different pre-plant treatments starting in summer 2002, including short-term fallowing (1 year when complete), short-term crop rotations (a summer crop of corn or sudan grass, or a fall/winter crop of wheat) and standard fumigation with MB/chloropicrin (50:50, 400 lb/A, imposed in November). Nemaguard peach seedlings will be planted in the micro plots in early 2003 and used to test for potential benefits of the pre-plant treatments under field conditions.

Cross specificity between peach and grape RD was studied in the greenhouse and is now being investigated in field micro plots. The greenhouse experiments did not provide conclusive evidence of such cross specificity, although in two of three tests, peach plants produced healthier roots in non-fumigated grape RD soil than in non-fumigated peach soil. Conversely, in the same two tests, grape plants produced healthier roots in non-fumigated peach RD soil than in non-fumigated grape soil, but the specificity was less pronounced for grape than for peach. For both crops, pre-plant fumigation with MB:chloropicrin (67:33) or autoclaving of the soil consistently increased growth (i.e., plant mass). A micro plot experiment was initiated near Parlier in 2002 to test for the cross specificity effects under field conditions.

Factors and organisms that cause or contribute to RD on Prunus species were investigated near Chico and Parlier, CA, using coordinated field, greenhouse, and lab experiments. At both locations, symptoms of RD in the experimental trees‚ shoots (i.e., growth cessation, wilting, or defoliation) appeared to result from poor root system development. Fewer healthy feeder roots were present on trees with RD symptoms in non-fumigated plots than on healthy trees in chloropicrin- or methyl-bromide fumigated plots. Isolations from the feeder roots on healthy and RD-affected trees revealed occasional association between root infection with Cylindrocarpon or Fusarium species and incidence of the disease. Greenhouse tests confirmed pathogenicity of these fungi, indicating that they can play at least a partial role in causing RD. Hundreds of bacteria were systematically isolated and preserved from the rhizospheres of healthy and RD- affected trees in the Chico and Parlier trials. The collection will facilitate future research to determine whether certain culturable bacteria play a significant role in RD. When semi-selective fungicidal and nematicidal treatments were imposed on RD soil in the greenhouse, one of the fungicides, but not the other chemicals, resulted in less severe root symptoms of RD on test plants (Nemaguard peach and Marianna 2624 plum), providing additional evidence for fungal involvement in the disease. In field experiments, chloropicrin, which is known for effective control of several soilborne diseases caused by fungi, was more effective in preventing RD than either 1,3-D or MB.

Continued work is needed on most aspects of this research. As the work progressed, it became apparent that the field environment is needed for full expression of RD. Therefore, micro plot studies were established at Parlier to augment the greenhouse experiments. Although the results indicate that some fungi not previously known as important pathogens of Prunus spp. contribute to RD, more work is needed to characterize them and their pathogenicity and to determine involvement of other microbes. Although this research accumulated an extensive collection of bacteria from the healthy and diseased trees, continued work is needed to characterize the sample populations and determine their effects on crop health. It is apparent that molecular approaches are needed to augment culture-based approaches to determining RD etiology, because most soil microbes are not culturable.

Specific Results

1. Determine effects of pre-plant bare-fallow periods and pre-plant cover cropping on development of replant disease (RD) on peach in California.

Determining effects of pre-plant fallow periods. Effects of pre-plant fallowing (0 to 3 years) on incidence and severity of RD were determined in four trials conducted by Tom Trout, USDA-ARS Water Management Research Laboratory (WMRL), Parlier. All of the trials were located on former sites of 10- to 20-year-old peach or plum orchards on USDA-ARS land near Parlier, CA.

The first two trials, replanted in 1998 and 1999, determined benefits of short-term pre-plant fallowing (0 or 1 year) for peach. Due to the conventional field planting schedules, the actual fallow treatments were 4 months (the “0-year” fallow treatment) and 16 months (the “1-year” fallow). For comparison, additional pre-plant treatments included: 1) methyl bromide (MB) (shank-injected, 350 lb/A under high-density polyethylene [HDPE]) with a 0-year fallow and 2) either Telone EC + Vapam (drip-applied 1,3-D, 310 lb/A + micro sprinkler-applied Vapam, 26 gal/A) or Inline + Vapam (drip-applied 1,3-D and chloropicrin mixture, 230 and 130 lb/A, respectively + micro sprinkler-applied Vapam, 13 gal/A), each with a 1-year fallow. The fallow treatments were imposed by removing trees in September at the required intervals before replanting (i.e., 0 or 1 yr). Within an experiment all replanting occurred on the same date (i.e., winter 1998 or 1999). The old trees were removed with conventional equipment that lifted and pushed out the root crowns and aboveground tree parts. Conventional ripping to 76 cm depth followed. Each pre-plant treatment had four replicate 19 x 16-m plots. Each plot was replanted with five five-tree rows of peach on Nemaguard peach rootstock. Tree performance was assessed annually by measuring the trunk diameters, pruning weights, and, when available, marketable fruit yield.

Two additional trials determined effects of 0- to 3-year fallows. For these experiments, old tree removal occurred 0 year (4 months actual) to 3 year (40 months actual) before replanting. All plots within an experiment were replanted at the same time in winter 2000. In both experiments, pre-plant fumigation with MB (shank-injected, 350 lb/A, under HDPE) served as a standard.

In the short-term fallow experiments, 1 year of fallow increased trunk growth on replanted trees during the first two growing seasons, compared to 0 year of fallow (Tables 1, 2). However, the growth benefit was not statistically significant in the third or later growing seasons, and it was not accompanied by significant increases in early marketable fruit yields (Tables 1, 2). The first-harvest marketable fruit yields following 0-year fallow treatments were 68 to 75% of those following pre-plant MB, while the first-harvest yields for 1-year fallows were 73 to 87% of the standard (Tables 1, 2). In the experiment planted in 1998, which accumulated three harvests by 2002, the yield advantage from fumigation eventually lost its statistical significance; the third-harvest yields for 0- and 1-year fallow treatments were 86 and 93% of the MB standard, respectively (Table 1).

In the long-term fallow experiments, increasing the length of pre-plant fallow periods from 0 to 3 year incrementally increased trunk growth and first-harvest fruit yields. In the first experiment, after 0, 1, 2, and 3 years of fallow, the respective yields were 40, 50, 70, and 108% of the MB standard (Table 3). Similarly, in the second experiment, after 0, 1, 2, and 3 years of fallow, the respective yields were 44, 57, 77, and 88% of the MB standard (Table 4).

The results of the fallow experiments indicate that at least 3 years of fallow are required to match growth and yield benefits provided by pre-plant fumigation with MB for the Parlier RD. Although 1 year of fallow did not provide large growth or yield improvements compared to 0 year of fallow, the benefits may be practically important, and the additional period before replanting allows growers more time for attending to physical and nutritional features of the replant site. It is unknown how well effects of fallowing in the Parlier trials represent benefits that would occur in other orchards. In the Parlier tests, the largest trees occasionally were pruned more heavily than would be typical for commercial practice, which could lessen the magnitude of potential treatment effects.

Determining effects of pre-plant cover crops. There is evidence that cultivation of a carefully chosen cover crop during the period between old orchard removal and new tree planting may help to manage RD. Cover crop growth and incorporation can improve a soil’s structure and increase its content of organic matter. Cover cropping can aggravate or suppress soilborne pests and diseases, depending on circumstances. Culture of certain wheat cultivars in replant soil from apple orchards suppressed development of apple RD in greenhouse tests (Mazzola and Gu, 2000).

Effects of pre-plant cover cropping were investigated in two greenhouse experiments. Soil samples from old peach and plum sites at Parlier were collected and planted to 10 different cover crops in 2-liter pots (Tables 5, 6). From 1 to 4 cover crop plants were planted per pot, depending on plant size. As controls, some of the soil samples were assigned bare fallow treatments. Fallow soil was kept either moist or dry, depending on the treatment, and some of it was fumigated with MB:chloropicrin (67:33, 0.8 g per liter of soil in a sealed 19-liter bucket) to serve as a standard. In each experiment, there were four replicate 2-liter pots of soil per pre-plant treatment. After 4 months of growth, the cover crops were shredded, incorporated into the soil, and allowed to decompose for 1 month. To assess effects of the pre-plant treatments on development of RD, 4-week-old Nemaguard peach seedlings were transplanted in the soils and grown for 3 months; three of the seedlings were planted in each replicate pot. At the end of the test, the seedlings were weighed, and the roots were washed free from the soil, rated for health, and subjected to fungal isolations.

In the greenhouse tests there was no clear, consistent effect of pre-plant cover cropping or fumigation on peach seedling growth or root infection by fungi (Tables 5-8). To test effects of pre-plant cover cropping under field conditions, micro plots were established in 2002 at the USDA center near Parlier. The micro plots were filled with soil from a nearby peach RD site and then subjected to one of several pre-plant treatments during the summer and fall. The treatments include short-term fallowing, short-term crop rotations (with corn, sudan grass, and/or wheat), and standard fumigation with MB/chloropicrin (50:50, 400 lb/A, imposed November 2002) (Table 9, Fig. 2). Nemaguard peach seedlings will be planted in the micro plots in early 2003, and performance of the plants will be used to assess effects of the pre-plant treatments.


2. Determine if cross-specificity exists between RD of peach and grape.

Because both peach and grape are affected by replant problems, a grower intending to plant peaches or a related crop on land previously devoted to grapes, or conversely, a grower shifting land from peach to grape would be concerned about the degree of specificity between peach and grape RD.

Transverse-planting experiments were conducted in a greenhouse. Soil was collected from 0 to 18” depths in three grape and three peach field sites at the USDA-ARS center near Parlier. All of the sites were within a half-mile radius. For Experiment 1, the soil was collected in early 1999 from replicate fumigated (MB, 350 lb/A, tarped, fall 1998) and non-fumigated plots in two areas, one previously devoted to peach and another to grape. The crops had been removed the previous fall, and the sampling was competed before replanting. Half of the soil from each background was autoclaved (70 min, 120 C) while the remainder was kept at room temperature until planting time in the greenhouse.

For Experiments 2 and 3, soil was collected in 2001 and 2002, respectively, and it originated from non-fumigated plots in old grape areas replanted to grape in 1998, 1999, or 2000, or non-fumigated plots in old peach areas replanted to peach in 1998, 1999, or 2000. Half of the soil from each background was fumigated in a sealed container (MB:chloropicrin 67:33, 0.3 g/liter of soil), and half was left non-fumigated in a sealed container as a control.

Depending on experiment, the soils prepared for Experiment 1, 2, or 3 were dispensed to 0.5- to 1.0-liter pots placed in a greenhouse, and each pot was planted with one Nemaguard peach seedling or one Carignane grape cutting or seedling. The plants were watered as needed (usually daily) and fertilized weekly with a liquid complete nutrient solution. Three months after transplanting, the plants were gently washed free from the soil. Top and root fresh weights and percent root discoloration were determined for each plant.

Results of Experiment 1 provided no conclusive evidence for cross specificity between peach and grape RD, but those of Experiments 2 and 3 provided some evidence for partial RD specificity. In Experiment 1, seedlings of peach and grape grown in non-fumigated, non-autoclaved soil developed significant amounts of root cortex necrosis, regardless of previous soil cropping history (Table 10). Either fumigating the soil before collection or autoclaving it after collection resulted in less root necrosis and more plant growth. In Experiments 2 and 3, relatively high percentages of root cortex necrosis occurred on grape and peach root systems grown in non-fumigated soil from grape and peach sites, respectively (Figs. 3-6, results organized to present the appropriate effects, based on analyses of variance). Comparatively less root damage occurred on peach when it was grown in non-fumigated grape soil instead of non-fumigated peach soil (Figs. 3, 4). The grape cuttings sustained moderate root damage in non-fumigated peach as well as non-fumigated grape soil, but severity of the damage was slightly less in peach than in grape soil (Fig. 5, results averaged for Experiments 2 and 3 due to lack of Experiment-by-treatment interaction). In Experiment 3, the response of Carignane grape seedlings to soil cropping history (Fig. 6) was similar to that of the cuttings (Fig. 5), except the seedlings developed more severe root damage. The container fumigation treatment consistently prevented root damage (Figs. 3-6) and generally, but not always, resulted in greater plant weights (Figs. 7-9). The plant weight data provided no clear evidence for RD host specificity (Figs. 7-9)

The results accumulated from greenhouse tests indicate it is not safe for growers to assume that there is no risk of RD when grape follows peach or vice versa. Because full expression of RD (and its specificity) may require a field environment, micro plots were established in spring 2002 to determine cross specificity effects in the field. Micro plots, each measuring 18 inches in diameter and 4 feet deep, were filled with soil from a vineyard or an adjacent peach orchard. There were 60 micro plots for each soil. Nemaguard peach seedlings were planted in the plots filled with the orchard soil, and Carignane grape cuttings were planted in those with vineyard soil. In spring 2003, the tops of the seedlings will be removed and the roots will be incorporated into the soil. Thirty of the micro plots for each soil source will be fumigated, and 30 will remain non-fumigated. Half the micro plots for each treatment history will be planted to grape, and the others will be planted to peach. Growth and health of the plants will be assessed and used to determine whether the RD exhibits host specificity.

Regardless of the experimental results in the greenhouse and micro plot tests, growers should keep in mind that some plant parasitic nematodes (i.e., ring and lesion nematodes), which, at infested sites, contribute to general replant problems in conjunction with RD, affect both grape and peach without apparent host specificity.

Determine organisms and factors that cause RD on selected Prunus spp. in California.

Approaching the problem. The approach for determining unknown causes of RD was: 1) to determine factors and symptoms associated with RD, 2) to determine microbes associated with RD symptoms, 3) to determine effects of general and semi-selective soil treatments on incidence and severity of RD, and 4) to test pathogenicity of microbes suspected to contribute to RD on Prunus species.

Characterizing RD in Chico area: associated factors, symptoms. For many years circumstantial evidence has suggested that a RD affects young almond trees in Butte County (Joe Connell, personal observations). First-year almond trees have sometimes failed to grow or died at high incidence after replanting at old almond orchard sites without pre-plant soil fumigation. Not all old almond sites have exhibited the problem. It is not known to occur when almonds are planted after field crops or after no crop. Affected trees are often on Marianna 2624 rootstock. Late planting dates or unsuitability of Marianna 2624 rootstock, rather than a RD, were initially suspected as causes of the orchard failures, so our experiments were designed to address these possibilities.

Almond replant experiments were conducted during 2001 and 2002 in a commercial orchard where more than 70% of the grower’s almond trees planted after removal of an old almond orchard died or failed to grow in 2000. The grower had not fumigated before replanting. In our experiments, rootstock and fumigation treatments were imposed to determine key factors and symptoms involved in the supposed RD. For each year of our tests, failed trees were removed in the fall (2000 or 2001) and replaced with experimental replant trees the following January (2001 or 2002), after imposition of selected pre-plant treatments (October 2000 or 2001).

Chico-area Experiment 1 tested effects of pre-plant broadcast treatments of MB (360 lb/A), chloropicrin (374 lb/A), Telone (360 lb/A), and a non-fumigated control, each imposed on four replicate 19- x 22-m replicate (18-tree-site) plots in randomized complete blocks. The fumigants were injected by shank on entire plots without plastic mulch.

Chico Experiments 2, 3, 4, and 5 determined effects of tree-site pre-plant fumigation treatments, rootstocks, scions, nursery propagation methods, and soil cropping history on incidence and severity of RD. The various treatment combinations, including fumigation, were assigned to individual or grouped tree sites. Depending on the experiment, there were four to nine replicate plots per treatment, with one to three trees per plot. All experiments had a randomized complete block design. A 2-ft.-diameter auger was used to prepare planting holes for fumigation and replanting. After soil was removed from the planting holes to a depth of 2 ft, it was used to refill the holes. This left the soil in a non-compacted state that was conducive to injection of fumigants at 18” depth (Figs. 10, 11).

All of the Chico field experiments involved monitoring tree performance for one growing season after replanting. Treatment effects were judged according survival and health of the replanted trees.

In Experiment 1, only the trees in the broadcast pre-plant chloropicrin plots performed well (Table 11). Relatively few trees planted after the control, methyl bromide or 1,3-D treatments grew acceptably. Chloropicrin has relatively broad biocidal activity at the rate that it was used, but it is best known for control of soilborne fungal diseases. The strong response to fumigation was evidence for a biologically mediated RD.

In Experiments 2 and 3, almond on Lovell peach rootstock was susceptible to RD, although less so than almond on Marianna 2624 (Tables 12, 13). French Prune on Marianna 2624 rootstock (known as a fully compatible graft combination) was as susceptible as almond on Marianna 2624, indicating that the Chico replant problem is not just a phenomenon of marginal graft compatibility between almond and Marianna 2624 (Table 14). Almond on Marianna 2624 trees were highly susceptible to RD, whether propagated commercially as potted plants or bare-root trees (Table 14). The fumigants 1,3-D, chloropicrin, and methyl bromide all were effective when applied to tree sites (Table 15), which contrasts to results with broadcast treatments in Experiment 1, where only chloropicrin was effective (Table 11). Tree site treatments are expected to result in higher localized doses of fumigant than broadcast treatments, even though at 1 lb per tree site they require only about 25 to 40% as much fumigant per orchard acre as common broadcast treatments. Filling planting holes with soil from an adjacent alfalfa field provided only partial control of the RD (Table 16).

In both years of the Chico tests, healthy and RD-affected trees (from chloropicrin-fumigated and non-fumigated plots, respectively) were compared to help characterize symptoms of the RD. Although all of the trees initiated shoot growth in the spring, those in the non-fumigated plots typically had ceased shoot growth by June, and they partially defoliated, wilted, and often died by July. When root systems were observed in May, healthy trees in the fumigated plots had produced new roots that generally had a whitish exterior, whereas the diseased trees produced fewer new rots, and many of them had become necrotic (brown, decayed) at the tips before growing or branching extensively. As the season progressed, many roots became dark on the outside, regardless of root health, and the most apparent difference between diseased and healthy root systems was that a much greater amount of fine roots was present on the healthy trees, compared to the diseased trees.

In greenhouse tests, Nemaguard peach seedlings and Marianna 2624 cuttings growing in non-fumigated soil from RD-affected areas consistently developed root cortex damage (Fig. 12). The symptom was not always accompanied by growth reduction, but it was reliably prevented by pre-plant fumigation of the soil in a container (0.3 g MB:chloropicrin 67:33/ liter of soil).

Our field and greenhouse observations suggest that aboveground RD symptoms (poor or lacking shoot growth) result, at least in part, from disease in the small roots. Inconsistent expression of shoot symptoms in the greenhouse suggested that the field environment is required for full development of RD.

Characterizing RD in Chico area: associated microbes. Isolations were conducted from the roots of healthy and RD-affected trees in Chico field trials (in chloropicrin-fumigated and non-fumigated plots, respectively). In 2001, the sampled chloropicrin plots received a broadcast treatment, whereas in 2002 they had received a tree-site treatment (1 lb per site). Root systems from four healthy and four diseased trees were excavated in May and August 2001 and May, July, and August 2002. The excavated roots were enclosed in plastic bags and kept cool for isolations. In the lab, fine roots (<1 mm diameter) were segregated, when possible, by health status (healthy=white exterior; diseased=dark and sunken exterior), and cut into segments (0.5 to 1 cm length), subjected to water rinsing or bleaching treatments (1 to 2 min, 10% commercial bleach, pH 7.0 to 7.2) and cultured on water agar (for general fungi; amended with tetracycline or ampicillin, 100 mg/liter), PARP (for oomycete “fungi”; corn meal agar amended with pimaricin, ampicillin, rifampicin, and PCNB). Eight to 16 (depending on the sampling date) water-rinsed and eight to 16 bleached root segments were cultured per medium per tree. Bacteria associated with the root surfaces and adhering soil were isolated by adding 0.2 to 0.4 g (depending on the sampling date) of the fine root segments to 5 to 10 ml sterile distilled water, respectively, vortexing for 1 min, and plating dilutions of the liquid on the following media: 10% Tryptic Soy agar (TSA, for general bacteria), RBME (for actinomycete bacteria), and MS agar (for oligotrophic bacteria). The root preparations were then subjected to bleaching for 1 to 2 min, rinsed in sterile distilled water, resuspended in 5 ml sterile distilled water, ground up with a Polytron PT 3000 (Kinematica, Lucerne, Switzerland), subjected to dilutions, and plated on the TSA, RBME, and MS media. The RBME and MS media were not effective in our tests and were abandoned after 2001.

Three days after plating roots on fungal isolation media, all fungi emanating from the roots were transferred from water agar and PARP plates to one-fifth strength PDA agar amended with tetracycline. Bacterial colonies on the bacterial media were counted after three days of incubation at 22 to 28 C. The fungal isolates were identified to genus according to morphology in 2001, but identifications and analyses are not yet complete for 2002 isolations. In 2001 and 2002, 50 to 60 randomly selected bacterial isolates per treatment (chloropicrin and non-fumigated) per sampling date (two dates in 2001 and two in 2002) were saved from 10% TSA cultures from Marianna 2624 rhizospheres for subsequent identification and pathogenicity tests. The bacterial isolates are preserved at -80 C.

Results of the 2001 fungal isolations from sampled roots suggest an association of root disease with species of Cylindrocarpon and Fusarium (Tables 17, 18). Several other fungi were isolated from the roots, but at relatively low frequency.

Soil was sampled to determine populations of plant parasitic nematodes in 2001 and 2002. The S.M. Schneider lab used the sugar flotation method to extract nematodes from soil samples collected around the roots of trees in experimental plots at depths of 0.5 to 2 ft. All plant parasitic nematodes present in the samples were identified to genus. Very few plant parasitic nematodes were detected in either year (Table 19), providing no evidence for their involvement in the RD at Chico. Root extractions will be completed in 2003 to compliment the sugar flotation extractions.

Characterizing RD in Chico area: effects of semi-selective soil treatments. A greenhouse test was conducted to determine if RD symptoms observed in the Chico field tests could be duplicated in the greenhouse, and, if so, whether certain organisms could be associated with the symptoms in the greenhouse tests. Soil was collected on 1/5/01 from non-fumigated and MB-fumigated plots of Chico Experiment 1. Each category of soil was mixed and split into two portions; one portion was autoclaved and the other remained at room temperature until testing. Soil from each combination of heating and fumigation treatments was further subdivided and randomly assigned treatments of difenoconazole (general fungicide, Dividend 3MG, 20 mg a.i./kg soil), fludioxonil (general fungicide, Maxim 4 FS, 20 mg ai/ kg), mefenoxam (oomycete fungicide, Ridomil Gold EC, 10 mg a.i./kg), streptomycin + chloramphenicol (bacterial antibiotics, each at 10 mg ai/kg), fenamiphos (nematicide, Nemacur 3, 30 mg ai/kg), or a non-treated control. The chemical amendments were added to soil by dissolving them in sterile water and spraying them into the soil samples with constant mixing; controls received sterile water alone. The treated soils were sealed in plastic bags and allowed to incubate at greenhouse temperature (19 to 25 C) for three days. The soil was distributed to 600-ml pots, and each pot was planted to a small Nemaguard peach or Marianna 2624 plant. At 1 and 2 months after transplanting, the difenoconazole, fludioxonil, and streptomycin + chloramphenicol treatments were repeated by drenching each pot with the required chemical in 100 ml of water, and the pots in control, mefenoxam, and fenamiphos treatments received 100 ml of water alone.

Among the treatment factors of field fumigation, autoclaving, and chemical amendment, only the latter two had significant effects on health of the roots on Marianna 2624 and Nemaguard assay plants (Table 20). Fludioxonil treatments significantly reduced the amount of root discoloration that occurred in non-autoclaved soil, compared to that in non-autoclaved soil with the other treatments. None of the other chemical amendments significantly affected root health. Autoclaving the soil prevented the root discoloration symptom. Chemical treatment had a negligible effect on top fresh weight of the plants (P=0.06), which was primarily due to slightly lower top fresh weights in the fludioxonil treated plants. Root weights were not affected significantly by chemical or heat treatments.

Root samples of Marianna 2624 and Nemaguard plants from selected treatments in the greenhouse test were cultured on media for isolation of fungi (water agar amended with tetracycline, PARP medium) and bacteria (10% TSA) using procedures described above (…Chico area: associated microbes). Incidence of Cylindrocarpon, Fusarium, Pythium, and Rhizoctonia exhibited close to marginal associations with symptoms of root disease (Tables 21, 22).

Characterizing RD in Parlier experiments, associated symptoms. For Parlier trials, RD symptoms were characterized on trees in the short and long-term fallow experiments (Determining effects of pre-plant fallow periods, above) as well as on additional trees in micro plots. For both sets of trees, the comparisons involved healthy and RD-affected trees in fumigated and non-fumigated plots, respectively.

The micro plots were established in spring 2002 as follows: concrete pipe sections measuring 4 ft tall x 18” diameter were installed into a field and then filled with soil from non-fumigated replant plots in one of T. Trout’s peach replant trials. On 4/30/02 pre-plant fumigation treatments were imposed on the soil in the micro plots and included a non-fumigated control, MB at 400 or 2700 lb/A, and chloropicrin at 400 or 2700 lb/A. Twelve replicate micro plots were allocated to each treatment. On 6/3/02 each micro plot was planted with three Nemaguard peach seedlings (2 months old from 2-inch pots, shoots trimmed to 3 to 4” length).

In the short and long term fallow tests, root systems were observed on a limited number of trees excavated by a backhoe. In the micro plots, shoot weights and root appearance were determined by sampling on 8/14, 9/24, and 11/20/02. On each date, shoot weights were determined and a coring tube (5-in.-diameter x 3-ft-tall.) was used to remove 5-in.-diameter x 18-in.-tall cylinder of soil and roots with the main root axis in the center (Fig. 13).

In the fallow experiments, root systems were much more massive in MB-treated plots than in non-fumigated plots. In the micro plots, shoot and root mass were several fold greater in plots that had been treated with chloropicrin at 400 or 2700 lb/A or MB at 2700 lb/A, compared to values from non-fumigated plots (Figs. 14,15; Table 23). The larger root systems had less root necrosis than the smaller ones (Fig. 15, Table 23). Shoots and roots in the 400 lb/A MB plots differed little from those in the control plots.

Characterizing RD in Parlier trials: associated microbes. Peach roots and the adhering rhizosphere soil were sampled from healthy trees in fumigated plots and RD-affected trees in non-fumigated plots on several occasions. At least four plots were sampled for each class of tree on each occasion of sampling. The sampled roots were subjected to bacterial and fungal isolations, using procedures similar to those described for Chico trials. For trees in short- and long-term fallow trials, root samples were collected from soil near the root crowns of first-leaf trees (6 to 18” depth). For the micro plots, root samples were obtained from the root cores described above. For both sets of trials, soil was collected around the roots for nematode quantifications.

In all three sets of fungal isolations from roots in the long and short term fallow trials, roots from non-fumigated plots had greater incidence of infection by species of Fusarium, compared to those from MB-fumigated plots (Tables 24-26). Cylindrocarpon was isolated more frequently from roots on diseased trees than from roots on healthy trees, but not consistently. Several other fungi were isolated, but none of them had an apparent association with lack of fumigation or root disease. Isolation results are not yet summarized for 2002 from plants in micro plot trials.

Bacteria were enumerated and preserved from two of the micro plot rhizosphere sampling episodes (120 isolates per treatment from non-fumigated, chloropicrin-fumigated (2700 lb/A) and MB-fumigated (2700 lb/A) treatments, but they have not been identified. They were streaked two times to obtain pure cultures and then preserved at -80C for subsequent testing.

Pin nematode counts exhibited some association with symptoms of RD in the fallow tests, but not in the in the micro plot study (Table 27). In the micro plots, severe RD symptoms were expressed in the apparent absence pin nematodes in micro plots given the low rate of MB (Tables 23 and 27). The simplest explanation for the result is that the low rate of MB was lethal to the pin nematodes but not to other biological agent(s) responsible for most of the RD symptoms. The result discounts the role of pin nematode in the peach RD at Parlier.

Characterizing RD in Parlier trials: effects of general and semi-selective soil treatments. In the first year of the project, it was determined that root cortex necrosis was induced on Nemaguard peach seedlings planted in non-fumigated soil from RD-affected Parlier sites. Pre-plant fumigation or dilution of the soil with fumigated soil prevented the symptoms and improved plant growth (Figs. 16, 17). In subsequent greenhouse tests, root cortex necrosis was not always accompanied by less plant growth.

Soil was collected on 7/27/01 from non-fumigated and fumigated 0-year fallow plots of the peach and plum long-term fallow tests near Parlier. One portion of soil from each field background was autoclaved, and the other remained at room temperature until testing. In the greenhouse, four Nemaguard peach seedlings were transplanted into 500-ml subsamples of soil (one seedling per 500-ml pot) for each combination of replicate field plot and soil autoclaving treatment; in total, 16 seedlings were allocated for each experiment-soil treatment background. The seedlings were watered and fertilized as needed (usually daily and weekly, respectively). Two months after transplanting, the seedlings were washed free from the soil and weighed. Root health was rated visually; those with dark-brown (necrotic) root cortex were rated as discolored. After seedling assessment, samples of the roots were cultured on water agar amended with tetracycline to detect fungi associated with them.

In each of the first two greenhouse trials, field fumigation or autoclaving the soil resulted in greater top fresh weight production by the Nemaguard seedlings, compared to the plants grown in non-fumigated, non-autoclaved soil (Tables 28, 29). The growth responses were associated with improved root health; 54 to 55 % of the roots in non-treated soil had discolored cortex tissue, whereas 5 to 39% of the roots in autoclaved or fumigated soil were diseased, depending on experiment and treatment. The isolations from the roots detected several different fungi, but their incidences did not clearly correspond to particular soil treatments (Table 30, 31).

In another greenhouse trial with autoclaved and non-autoclaved soil from the Parlier RD trials, the soil was subjected to a series of semi-selective chemical treatments. The chemical treatments included: difenoconozole (Dividend 3MG, 20 mg ai per kg soil), fludioxonil (Maxim 4FS 20 mg ai per kg), and mefenoxam (Ridomil Gold EC, 20 mg ai per kg), fenamiphos (Nemacur 3, 15 mg per kg), chloramphenicol (50 mg per kg), streptomycin (80 mg/ kg), and chloramphenicol + streptomycin (50 + 80 mg per kg). The chemical treatments were applied only once, 3 days before planting by atomization into constantly stirring soil samples. Four replicate Nemaguard peach seedlings were used to assay effects of each soil treatment in one greenhouse experiment. Planting culture and disease assessment were as described for the greenhouse experiments above. Roots of the plants were cultured on water agar at the end of the tests (10 roots per replicate plant) to determine if certain fungi were associated with root disease symptoms.

Among non-autoclaved soil treatments other than the one that received fludioxonil, an average of 60 to 76% of the peach roots were discolored at the end of the test, indicating disease (Table 32). Either pre-plant soil autoclaving or the treatment with fludixonil reduced the percentage of diseased roots (to 14 to 39%). Pre-plant soil autoclaving, but not pre-plant fludioxonil treatment, reduced incidence of Fusarium spp (Table 33). Other fungi occurred at very low incidence, regardless of treatment.

Pathogenicity tests. Thirty-five representative isolates of fungi from necrotic roots of RD-affected Parlier trees were tested for pathogenicity in a greenhouse. Inoculum of each isolate was grown on an oat bran substrate and mixed with autoclaved Yolo Loam soil a rate of 0.4% (by weight, moist inoculum and air-dry soil). Nemaguard peach seedlings (4 weeks old) were exposed to each fungus by transplanting them in 0.6-liter pots with the infested soil preparations (one seedling per pot). Control seedlings were planted in non-infested soil (amended with sterile oat bran substrate, 0.4%). Additional seedlings were planted in soil amended with autoclave-killed inoculum of Fusarium oxysporum (0.4%), a mixture of putatively non-pathogenic fungi, and a mixture of putatively pathogenic fungi (Tables 34, 35). Each isolate was represented equally in multiple-isolate mixtures, and mass of the mixed inocula totaled 0.4% of the soil mass. Ten replicate peach seedlings were used per soil treatment, randomized in a complete block design. After transplanting, the seedlings were watered daily and fertilized 1 to 2 times per week.

Seven weeks after transplanting, the plants were assessed for disease by determining height, fresh weights, and amounts of root necrosis for each seedling. Significant growth reductions (i.e., reduced top or root weight) and/or root necrosis consistently were caused by isolates of Cylindrocladiella peruviana, Fusarium spp., Humicola grisea, Pythium sp., and Trichoderma sp., compared to the non-inoculated control (Tables 34, 35). One of the Cylindrocarpon isolates was pathogenic in the first pathogenicity test (Table 34), but all were pathogenic in the second (Table 35) and subsequent tests (data not shown). All of the pathogenic isolates were reisolated from the symptomatic roots (data not shown), thereby fulfilling Koch’s postulates for the fungi that showed association with symptoms of root disease in the field (i.e., Cylindrocarpon sp., Fusarium spp.) Seedlings grown in soil with the “pathogen mixture” or the “all fungi mixture” produced less growth and had significant amounts of root necrosis, compared to the control (Tables 34, 35). The “non-pathogen” mixture reduced growth of the seedlings but did not cause root necrosis. The disease caused by Trichoderma sp. was not expected (it is not known as a pathogen of Prunus spp.) and may reflect a degree of artificiality in the screen for pathogenicity.

The results of our field and greenhouse isolations combined with those of our pathogenicity tests to date indicate that at least some species of Fusarium contribute to RD on Prunus spp. A role of other fungi, (i.e., Cylindrocarpon sp., H. grisea, and Pythium sp.) seems likely due to their demonstrated pathogenicity on roots but remains uncertain due to their infrequent isolation from diseased roots.

Continued work is needed on most aspects of our research. As the project progressed, it became apparent that a field environment is needed for full expression of RD. This realization required us to spend more time than expected to set up field trials and micro plots. Although our research indicated that certain fungi may contribute to Prunus RD, it still is uncertain whether they are the primary causes of the disease. Molecular approaches are needed to augment culture-based approaches to determining RD etiology, because many soil microbes are not culturable. Although our research accumulated an extensive collection of bacteria from the healthy and diseased trees, continued work is needed to characterize the populations and determine isolate effects on crop health.

Potential Benefits/Impacts on Agriculture

U.S production and importation of MB will be curtailed in 2005, with possible exceptions for quarantine pre-shipment treatments and critical use exemptions. The fumigant currently provides for critical management of known and unknown soilborne pests and pathogens, including cause(s) of RD on Prunus spp. Continued competitive production of these crops, whether in nursery or orchard settings, will require cost-effective MB alternatives.

Our finding that 1-year fallow period may provide only partial control of RD indicates the importance of determining how to augment such effects with other practices. For most growers, imposition of the 3-year or longer pre-plant fallow periods required for complete RD control is not economically feasible.

Results of our greenhouse tests provided no evidence for cover crop suppression of RD or cross specificity between peach and grape RD. Nevertheless, the greenhouse results provided the impetus for establishing micro plots to determine cover crop effects. We now consider it is likely that the field environment is needed for effective testing of cover cropping and cross-specificity effects. Field micro plot experiments on RD were established in 2002 and are anticipated to provide stone fruit and nut growers with valuable field-derived data on effects of: 1) short-term fallowing or rotations with corn, sudan, wheat, or sudan followed by wheat, and 2) cross specificity between peach and grape RD.

Our research on unknown causes of RD has focused on involvement of culturable fungi, although a foundation collection was established to facilitate similar work with culturable bacteria. Our results indicate that certain Fusarium and Cylindrocarpon species can contribute to RD, but continued research on RD etiology is needed and underway. Our Chico and Parlier field trials have demonstrated that RD can occur in the absence of significant soil populations of plant parasitic nematodes. Growers often use plant parasitic nematode counts to indicate risk of replant problems, but the results reported here indicate that the information does not predict risk of the RD component of the problems.

Practical side benefits resulted from the research on RD etiology. The research revealed that chloropicrin is a particularly potent MB alternative for prevention of RD. In addition, tree-site fumigation with an appropriate fumigant was found to prevent RD. Although the focused tree-site treatments would not be effective where there are high populations of plant parasitic nematodes, they were sufficient for control of RD in Chico tests. Tree site fumigation at 1 lb per tree site requires only about 25 to 40% as much fumigant per orchard acre as common broadcast fumigation. This significant reduction in fumigant requirement could save growers money as well as reduce environmental hazards. Tree site treatments merit further research.

Dissemination of Findings

Oral presentations of this research were made at the following meetings:


Publications and reports from this research:


Literature Cited

Mazzola, M. & Y.-H. Gu. 2000. Impact of wheat cultivation on microbial communities from replant soils and apple growth in greenhouse trials. Phytopathology 90: 114-119.

 

Home | Search | Calendar | Contact Us | Webmaster | Copyright Notices