EPIDEMIOLOGY AND MANAGEMENT OF BOTRYOSPHAERIA/PHOMOPSIS

CANKER AND BLIGHT AND ANTHRACNOSE BLIGHT OF WALNUT IN

CALIFORNIA

Themis Michailides, David P. Morgan, Daniel G. Felts, Yong Luo, Ryan Puckett, Janine Hasey,
Bruce Lampinen, Emily Symmes, William Coates, Richard Buchner, Dani Lightle, Elizabeth
Fichtner, David Rodriguez, and Connor Cunningham

ABSTRACT
The Botryosphaeriaceae fungi and Phomopsis species are now widely distributed in walnut
orchards in all the counties where walnuts are grown. In fact, 20% of the samples were diagnosed
infected by Botryosphaeria, 20% infected by Phomopsis, and the majority of the remaining
samples had both Botryosphaeria and Phomopsis together while only a few of the samples were
diagnosed as branch wilt caused by Neoscytalidium dimidiatum (the former branch wilt fungus
Hendersonula toruloidea). Furthermore, in some cases the branch wilt fungus killed entire 1 to 2
year old trees in the field. Again, in 2016, for both Botryosphaeria and Phomopsis, we confirmed
the presence of both reproductive structures, pycnidia producing the water-splashed conidia and
perithecia producing the airborne ascospores. This information is of major importance because it
helps us understand how these fungal pathogens spread within and among the walnut orchards and
among other susceptible agricultural and riparian tree and bush species. Although conventional
methods of trapping the spores of these fungal pathogens under drought conditions are not
sensitive enough to detect low amounts of released spores, the developed effective and very
sensitive quantitative PCR method (qPCR) can detect low amounts of spores trapped with spore
traps after a rain event. Spores were detected for the major fungi causing Bot canker and blight in
a walnut orchard from May through mid-September. An experiment simulating a rain of 8 hours
showed that the majority of pycnidiospores of Neofusicoccum are released within the first 2 hours
after the initiation of the rain and the pycnidia are exhausted of the majority of the spores within
the first 4 hours of the rain. Pruning wounds of walnut remain susceptible to infection by the
Botryosphaeriaceae fungi for at least 4 months after the wounds are made. Shoots of 3- to 4- years-
old wood are more susceptible and developed larger cankers than those of 1- to 2-years-old shoots
(confirmed for a 2nd year). The majority of fungicides used on cuts immediately after pruning did
not protect the wounds from infection. Only Merivon significantly reduced the percentage of
cankers in shoots pruned and inoculated with Lasiodiplodia citricola in October 2015 (fall
pruning); this effect was not seen in February 2015 prunings (winter pruning) or with
Neofusicoccum parvum inoculations at both pruning dates. In fungicide spray experiments, all of
the treatments with Merivon at various timings significantly reduced blighted fruit. The best
Merivon® treatment was four sprays at bloom (31 March), on 5 May, 10 June, and 12 July. Among
a number of treatments with different timings with Merivon®, the calendar-scheduled program
(conventional), the timing Experiment, and the spray program predicated by the Leaf Wetness
Model (LWM) proved most effective. The LWM triggered 3 sprays each done before or
immediately after an infection event (at least 50°F and at least ¼ of an inch wetness). We also
provide evidence again that the majority of the fungicide sprays applied in spring and summer
have a long-term effect (approximately 6 to 8 months after application) on reducing
Botryosphaeria canker and blight significantly as determined by the pre-season BUDMON assay.
Three sprays in the spring (5 April to 1 June) of a number of fungicides showed efficacy against

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the anthracnose of walnut (second year efficacy data against this disease). Interestingly and to our
surprise, a few specific fungicides and combinations of fungicides with biologicals (see Table 5
for details) reduced significantly walnut blight similarly to the standard grower spray with Badge
+ Manzate.

OBJECTIVES
1. To continue surveying walnut orchards and diagnosing diseased samples brought/sent to the
laboratory by growers, farm advisors, pest control advisers, and other industry representatives.
2. To monitor the dynamics of inoculum over the seasons in walnut orchards.
3. To investigate latent (symptomless) infection of buds, shoots and fruit as a tool to predict
disease risk in orchards.
4. To determine the relationship of pruning wounds to susceptibility to infection.
5. To determine the efficacy and best timing of fungicides to control Botryosphaeria and
anthracnose blights in walnut.

SIGNIFICANT FINDINGS
1. Confirm the presence of two types of inoculum (water-splashed conidia and air-borne
ascospores) produced by Botryosphaeria and Phomopsis species in walnut orchard.
2. The quantitative PCR method detected Phomopsis only in April, while Botryosphaeria
dothidea, Lasiodiplodia, Cytospora and Neofusicoccum spp. from April to
August/September; and Diplodia spp. were found only sporadically.
3. Higher incidence and greater severity of cankers developed on spurs after fruit
inoculation with Lasiodiplodia citricola but not with Neofusicoccum parvum with late-
season inoculations than early –season inoculations (linear relationship).
4. Newly-emerged (spurs)/ shoots harbor latent infection by Phomopsis, Botryosphaeria
dothidea, and species of Lasiodiplodia, Neofusicoccum, and Cytospora, but not Diplodia.
The results were confirmed with the quantitative qPCR assay,
5. The qPCR worked well and detected the latent infections on fruit. The molecular severity
levels corresponded well with the incidence of latent infection on fruit, newly-developed
spurs, and 1-year-old shoots.
6. Initially the 1-year-old shoots had higher levels of latent infection, but this difference
diminished as the season progressed.
7. We confirmed that pruning wounds are susceptible for at least 4 months, and the wounds
of 3- and 4-year-old shoots favor the development of larger cankers than the 1- and 2-
year-old shoots.
8. Treating pruning wounds created in February (winter pruning) and inoculating with
Lasiodiplodia citricola 1 day after fungicide treatment was not effective in reducing
either infection or canker length.
9. For the fall (29 Oct) pruning though, the fungicide Merivon protected the pruning wounds
from infection.
10. Several fungicide calendar sprays done on 6 May, 10 June, 12 July, and 2 August reduced
significantly the incidence of infected fruit and cankered spurs (see Table 1 for details).

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 11. We confirmed that a bloom spray (31 March) reduces Botryosphaeria/Phomopsis canker
and blight.
12. Sprays in June, July, or August with Merivon provided numerically the best control.
13. The leaf wetness model (LWM) predicated 3 sprays (26 April, 23 May, and 20 June)
provided similar control as the 4 calendar sprays.
14. Several fungicides applied at the ½ leaf size (April 5) to 1 June (3 total sprays) provided
a second year good efficacy results against anthracnose of walnut.
15. Surprisingly, Luna Experience + Serenade Opti(!), the unregistered Kenja(!) (isofetamid)
fungicide, Luna Experience + Movento applied on 5 April, 28 April, and 1 June
controlled walnut blight equally well as the standard treatment Badge + Manzate applied
on the same dates.
16. Other fungicide treatments were less effective but showed significantly lower walnut
blight than the untreated control, suggesting that sprays done against Botryosphaeria
canker and blight also help to reduce walnut blight.
17. We confirmed that the majority of the fungicide treatments have a long term effect in
reducing Botryosphaeria in walnut buds and some of them also reduce Phomopsis.

PROCEDURES

1) To continue the survey of walnut orchards and diagnose diseased samples brought/sent to
the laboratory by growers, farm advisors, and pest control advisers.

We continued to diagnose samples brought to the laboratory by walnut growers, farm advisors,
pest control advisers, and representatives of various chemical companies. We like this activity
because we feel we provide walnut growers with direct and timely help, and advice on the
diagnosis of disease in their walnut orchards, giving them the opportunity to design and/or adjust
pest management approaches accordingly. Diagnosis is done “on the spot”, either by direct
observation, examining spurs for the presence of pycnidia, by plating the margins of cankered
tissues, or by plating samples of random buds in early spring by using the BUDMON Technique.
Diagnostic results are communicated back to the interested parties either by a phone call or mainly
by an e-mail message so that the grower has also a record of the diagnostic results.
2) To monitor the dynamics of inoculum over the seasons in walnut orchards.
In this year, we continuously focused on identification of canker-causing pathogens and their
corresponding quantities as inoculum in the air. We quantified the dynamics of the inoculum in a
Chandler walnut orchard with history of severe disease found immediately next to the Sacramento
River. We developed molecular approaches by using real-time (quantitative) PCR (qPCR) to by
using SYBR method to identify and distinguish six canker-causing fungal species, including
Phomopsis spp., Botryosphaeria dothidea, Lasiodiplodia spp, Cytospora spp., Neofusicoccum
spp., and Diplodia spp (Luo et al, 2017). The standard curves from this study were used to quantify
the inoculum in the air and to quantify latent infection levels of walnut shoots as follows.
Two Burkard spore traps (Cyclone sampler for field operation (Burkard Manufacturing Co. Ltd,
Rickmansworth, Hertfordshire, UK) were placed in two different areas (namely East and West) in
a walnut orchard located next to Sacramento River in Butte County of California. The air samples
were collected approximately every 3 to 4 weeks into a 1.6 ml centrifuge tube. The velocity of the

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air during operation of the trap was approximately 11520 L of air/day. One ml of distilled water
was added in each sample tube and processed as described below.
The spore trap samples were processed in 2016 by using the sensitive qPCR method developed in
this study. For each spore trap sample, 200 µl of each of the original spore suspension was used to
extract DNA, and the qPCR systems were applied to quantify the number of spores as log 10
(number of spores/day) for each pathogen.

3) To investigate latent (symptomless) infection of buds, shoots and fruit as a tool to predict
disease risk in orchards.
Latent infection of walnut fruit. To determine when hulls are infected, first immature and then
maturing Vina fruit were inoculated in the field with Lasiodiplodia citricola (isol. 6-I35) and
Neofusicoccum parvum (isol. 1-L87), using 7 fruit per isolate on each of 3 trees. Inoculations were
done every 3 weeks, beginning May 12, 2015 with the last inoculation done Sept 15, 2015. Shoots
with fruit were sprayed with a 15,000-50,000 spores/ml suspension of Lasiodiplodia citricola or
Neofusicoccum
parvum. The inoculations were performed in late afternoon, bagged with a plastic bag to maintain
humidity, and covered in a white paper bag to prevent sunburn and overheating. Bags were
removed early the following morning. Inoculated fruit from the first five inoculation dates were
collected Sept 24, 2015; fruit from the last two inoculations were collected on Oct 26, 2015.
Blighted fruit were collected and recorded for each treatment and isolations made to determine if
the pathogen used for inoculation could be recovered. The results from the fruit were presented in
last year’s report. We collected these shoots March 22, 2016 and measured cankers that had
developed after inoculation.
Similar inoculations were made May through September of 2016. Pathogens used this year were
Lasiodiplodia citricola, Neofusicoccum parvum, and Phomopsis sp. The number of inoculations
was increased, with 10 fruit clusters on each of three trees for each pathogen. Fruit were removed
from the clusters in early November but isolations have not been done. The shoots will be collected
in March for canker measurement.
Quantification of latent infection level of shoots and fruit using molecular approach.
In the same orchard used in Objective 2, shoots were collected periodically in April, July and
October of 2016. For each sampling, about 10-20 cm-long shoots, including the newly-emerged
shoots and the top portion of the old shoot in the proximity of the new shoot, (usually 1-year old)
were collected. Thus, each sample contained two parts: new shoots and old shoots, and processed
separately. For each sampling, 32 such shoots were randomly collected. These shoots were washed
twice with tap water, soaked in 10% commercial bleach for 10 min for surface sterilization, washed
three times again, and air dried for 2 days. A pencil sharpener was used to grind shoot samples
into fine wood pieces which were used to extract DNA by using the FastDNA kit (MP Biomedical,
CA). Briefly, the pathogen group-specific primers were used in real-time PCR to obtain the
corresponding Ct values. The published equation of standard curve for each pathogen group (Luo
et al., 2017) was used to calculate the DNA quantity for each pathogen in each sample.
To quantify the infection level of shoots, we introduced the concept of molecular severity (MS):
MS= Log 10 (P/H), where P is the weight of the pathogen’s DNA in femtograms (fg), which is

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calculated by using the equation of the standard curve for the corresponding pathogen (Luo et al.,
2017) based on the Ct value from its reaction with the corresponding primers, and where H is the
shoot weight in grams (g). Thus, if the minimum detectable amount of pathogen DNA in one gram
of shoot is theoretically assigned as 1 fg, the MS would be 0. The maximum of the amount of
pathogen DNA in one gram of shoot tissue could be theoretically one gram (=1015 fg), and the
maximum value of MS should be 15. Thus, the range of MS value is 0 - 15. However, since when
no infection is detected we assign MS = 0, the theoretically detectable amount of pathogen DNA
in one gram of shoot should be >1fg. The concept and calculation of MS were used to determine
the infection level for all the shoot samples used in this study.
The incidence of latent infection in terms of proportion of shoots showing positive results over all
detected shoots was obtained for each pathogen on each sampling date. Average MS from 32 shoot
samples was obtained for each pathogen and each sampling date. Comparisons in MS between
new and old shoots were conducted for each pathogen and on each sampling date.
Similarly, latent infection levels in fruit were determined in May of 2016 for the orchard in Butte
Co. mentioned above and the walnut orchard in KARE. In early May of 2016, 30 fruit and 24 fruit
were randomly collected from the walnut orchards in Butte Co. and KARE, respectively. The same
approaches using qPCR assay mentioned above were applied. The incidences of latent infection
and molecular severities (MS) for both orchards were quantified.
4) To determine susceptibility of pruning wounds to infection.
To determine whether and how long pruning wounds are susceptible to infection, one of the most
aggressive (virulent) species, Lasiodiplodia citricola (isol. 6-I35) was used for this experiment.
The Chandler walnut cultivar at Kearney experimental orchard was pruned as follows: Groups of
10 each from 1-, 2-, 3-, and 4-year-old shoots were pruned and inoculated. Inoculations with a
spore suspension of 50,000 spores/ml were done with post-pruning intervals of 0 days, 3 days, and
1, 2, 3, 4, 8, 12, and 16 weeks. This experiment began on 9 February 2015. Ten non-inoculated
pruned shoots from each age category served as the control treatment. Shoots were collected and
cankers measured March 7-10, 2016. (Note: 10 shoots × 9 inoculation dates × 4 age categories +
40 control shoots = a total of 400 shoots were used in this experiment.)
The pruning and inoculation procedure was carried out twice in 2015. The second experiment
began on 27 October 2015 and the inoculation schedule was as described for the February pruning.
Cankers were measured after collecting the shoots. Isolations were taken from fall pruning cankers
and the cankers were only counted if the inoculated pathogen was recovered. Isolations were not
taken from the spring pruning. Canker lengths were recorded but results are presented in relation
to the percentage of inoculations that resulted in canker formation.

5) To manage Botryosphaeria canker and blight and anthracnose blight in walnut (fungicide
trials).

Protecting pruning wounds: Potential fungicides used to protect pruning wounds of walnuts were
tested for their efficacy against infection by Botryosphaeriaceae. Chandler trees in our
experimental orchard at Kearney were used in this experiment. Shoots of age 1-, 2-, 3-, and 4-
years were pruned as described in objective #4 above. Immediately after pruning, wounds were
treated with one of the following fungicides: Merivon, Merivon + VitiSeal, Quadris Top, or

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Abound. Five shoots of each age were used per treatment, for a total of 200 shoots pruned on Feb
12, 2015, then treated with fungicide, except for 10 shoots in each age category which served as
controls. The day following the treatments, 5 shoots in each group were inoculated with
Lasiodiplodia and 5 with Neofusicoccum, including the untreated control shoots. Cankers were
measured on 7 March 2016.
The Merivon and the control treatments as described above were repeated to compare spring and
fall pruning times. Pruning was done on 29 Oct and inoculation on 30 Oct 2015. Cankers were
measured on 17 November 2016. Canker lengths were recorded but results are presented in
relation to the percentage of inoculations that resulted in canker formation.
Fungicide concentrations were based on applying the field rate of oz/acre in 5 gallons of water per
acre. These rates were as follows: Merivon = 6.8 oz/acre; Quadris Top = 14 oz/acre; Abound =
12 oz/acre; Viti Seal 1:10 dilution of the commercial product.
Fungicide trial in Butte County. We established a fungicide trial in Butte Co. in 2016. A Chandler
orchard located adjacent to the Sacramento River which had a history of severe infection in 2014
was used. The following experiments were done in this orchard: a) a trial for the efficacy of various
fungicide treatments based on a calendar spray; b) a trial with sprays based on the Bot infection
model (Morgan et al., 2010); c) a trial to determine the “best timing” by spraying Merivon only
once at different dates to different sets of 5 trees; and d) a trial to compare efficacy of Merivon
bloom spray only with the Merivon postharvest spray only to determine whether such sprays
(bloom or postharvest) have any efficacy against reducing the disease. All the trees used for the
above fungicide trials were randomized in this orchard using 5 replications in 5 different rows of
trees. All the fungicides were sprayed with a handgun using our 4-tank sprayer. Fruit with black
hulls were counted on the ground after commercial shaking, on 28 September 2016, the day of
harvest. The incidence of blighted current season shoots was recorded using pruning towers on 5
and 6 December 2016. Furthermore, 25 buds per tree (a total of 125 buds per treatment) will be
collected in Feb/March 2016 to perform the BUDMON assay and determine the long effect of
fungicides on infestation of buds by species of Botryosphaeriaceae and/or infection by Phomopsis.
Fungicide trial in Yuba County. This trial was set up in a Vina orchard in cooperation with
cooperative extension farm advisor Janine Hasey. Merivon was sprayed at 6.5 fl oz/A with 3 fl oz
/A organic silicone at 100 gallons/acre at various timings. There were four replicates consisting
of entire tree rows with buffer rows between treated rows. All the fungicides sprays were done
using the grower’s commercial rig. Buds for the BUDMON assay were collected in February 2016
to determine the effects of fungicides (Fig. 21-22).
Fungicide trial in Colusa County. This trial was set up in a Chandler orchard in cooperation with
farm advisor Janine Hasey. The fungicides were sprayed using the grower’s commercial rig. There
were four replicates consisting of entire tree rows with buffer rows between treated rows. Buds
for the BUDMON assay were collected in February 2016 to determine the effects of fungicides
(Fig. 23-24).
Fungicide trial in Colusa County—Nickels Soil Lab. This trial was set up in a Chandler orchard at
Nickels Soil Lab in cooperation with farm advisor Janine Hasey and specialist Bruce Lampinen.
The fungicides were hand sprayed to run-off on pruning wounds using spray bottles. Primary
comparisons in this experiment are a hedging trial vs. a whisking (lateral limbs removed on north
and south sides, ~10 cuts/tree) trial, both pruned on 28 March 2016. In the hedging trial there are

California Walnut Board 6 Walnut Research Reports 2016

five prunings per treatment in each of six reps. In the whisking trial there are three prunings per
treatment in each of four reps. The treatments are: 1) untreated control; 2) Quadris Top, 14 fl
oz/ac; 3); Quadris Top + Viti-Seal, 14 fl oz/ac; 1:50; 4) Quadris Top + Spur Shield, 14 fl oz/ac;
1:50; 5) Tilt, 8 fl oz/ac; 6) Tilt +Viti-Seal, 8 fl oz/ac; 1:50; 7) Tilt + Spur Shield, 8 fl oz/ac; 1:50.
One year after treatment, cankers will be measured on treatment and control limbs and samples
taken to identify Botryosphaeria and Phomopsis species present on dead limbs in the orchard.
Fungicide trial against anthracnose in San Benito County. The walnut anthracnose disease, caused
by the fungus Gnomonia leptostyla, the perfect stage of the fungus Marssonina juglandis, has
created some problems in walnut cultivars in commercial orchards and walnut rootstock (i.e., black
walnut) in nurseries in the past under normal, yearly rainfall. Because the anthracnose disease was
very severe in walnut orchards in San Benito County in 2011 and 2012, we easily found both the
conidia and the ascosporic stages of the fungus in an orchard which the grower allowed us to use
for a fungicide trial. The conidia are produced in saucer-like structures produced on leaves, hulls
of fruit, petioles, and shoots. The perithecia (which produce the airborne ascospores of the fungus)
were found in leaves on the ground in the spring. When the perithecia become wet from sprinkler
irrigation or rain, the ascospores are shot into the air and become airborne. In 2016, we established
a fungicide efficacy trial. The fungicides used, the label rates, and dates of sprays are listed in
Table 5. Because of the many treatments we had to use in 2016, we reduced the number of 4
replicated trees to 3 trees per fungicide treatment.
Residual activity in 2016 of fungicides sprayed in the spring of 2015. The long term activity of
fungicides against Botryosphaeriaceae species was studied because previous research showed that
fungicide sprays resulted in reduction of primary inoculum of apple powdery mildew during the
season following the application season. We evaluated the level of bud infection/infestation by
Botryosphaeria and/or Phomopsis during the late winter of 2016 about 8 months from the time the
last spray was applied on to the trees in 2015. Fifty dormant buds were collected on five 2015
fungicide trials in walnut orchards located in Butte (one south of Chico, and another east of
Durham) a third in Colusa, a fourth in Yuba, and a fifth in San Benito counties. Buds were plated
on acidified PDA according to the BUDMON technique and plates incubated at 27ºC (81ºF). The
incidences of Botryosphaeria/ Phomopsis were recorded 7 days after plating and incubating the
plates.

RESULTS AND DISCUSSION

1) To continue the diagnosis of diseased samples brought or sent to the laboratory by

growers, farm advisors, pest control advisers, and industry representatives.

We diagnosed 34 walnut samples in 2016. Of these, 7 samples produced species of

Botryosphaeriaceae and 7 were found to have Phomopsis species; some of these samples had both
pathogens. The majority of these samples had pycnidia of these fungi, fruiting structures produced
in infected plant tissues underneath the epidermal layer producing pycnidiospores. Pycnidiospores
require water to exude from the pycnidia and spread to infect plant tissues. In four of these 2015
samples that involved blighted shoots at least 1 year old (3 from Butte Co and one from Tulare
Co.), in addition to the pycnidia of Botryosphaeria and Phomopsis, we also found perithecia
produced by these fungi. Perithecia are fruiting structures that produce asci (little sacks) with 8
ascospores each. The importance of this type of spores is that when the perithecia get wet (i.e.

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from rains or sprinkler irrigation), the ascospores are forcibly ejected into the air and become
airborne. In previous studies we also determined perithecia in walnut branches collected from the
ground in commercial orchards in Stanislaus, Butte, Colusa, and Sutter Counties and in shoots
collected from trees. The perfect state of Phomopsis species belongs in the genus Diaporthe. At
least two species of Diaporthe (D. rhushicola and D. neotheicola) isolated from walnuts were
identified (Chen et al., 2014). Therefore, both species of Botryosphaeriaceae and the Phomopsis
species produce both pycnidiospores and ascospores in walnut. This is important information
because it helps to realize how these fungal pathogens spread among walnut orchards. The
diagnosis of each sample was communicated on time to farm advisors, pest control advisers, or
growers who submitted samples in 2016.
2) To monitor the dynamics of inoculum during the season in walnut orchards.
The six pathogen groups were all found during the season, while Diplodia spp. was detected
sporadically at very low level (Figure 1). The species of Phomopsis was detected only in the early
stage in the orchard and not found later in the season (Fig. 1). In the air, B, dothidea, Lasiodiplodia
app. Cytospora spp. and Neofusicoccum spp. were the dominant pathogens in the orchard. These
four pathogen groups were detected in most sampling periods, indicating the major component of
canker-causing pathogens in walnut orchard. Generally, the quantities of B. dothidea were higher
than those of other pathogens in different sampling periods. The spore densities of these pathogens
ranged from several tens to several hundreds of spores per day (log 10 (no. spores/day) = 1 to 2.5).
Although Cytospora is not reported as a pathogen of walnut in California, there were a lot of spores
of this pathogens collected by the traps in the orchard. This is most likely spore inoculum produced
in susceptible hosts, such as prunes, almonds, and riparian tree species that are abound in the
vicinity where the walnut orchard is located.

California Walnut Board 8 Walnut Research Reports 2016

 3.00 3.00
2.50 Phomopsis spp. 2.50 Botryosphaeria dothidea
2.00 2.00
1.50 1.50
1.00 1.00
0.50 0.50
0.00 0.00
4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29
Log10 (no. of spores/day)

4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29

3.00 3.00
2.50 Lasiodiplodia spp. 2.50 Cytospora spp.
2.00 2.00
1.50 1.50
1.00 1.00
0.50 0.50
0.00 0.00
4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29 4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29

3.00 3.00
2.50 Neofusicoccum spp. 2.50 Diplodia spp.
2.00 2.00
1.50 1.50
1.00 1.00
0.50 0.50
0.00 0.00
4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29 4/7 - 4/26 4/26 - 6/9 6/9 - 7/12 7/12 - 8/2 8/2 - 9/29

East West East West

Period of time in 2016

Figure 1. Spore densities of six canker-causing pathogens in air samples collected with two Burkhart spore
traps (East trap and West trap) installed in a Chandler walnut orchard in Butte Co. Spore densities were
quantified with the qPCR method developed in this study. Five samplings were conducted, each
representing and containing the total spores in the air collected during a period of time in 2016. The specific
primer pairs were used to quantify the density of spores of the corresponding species in the same samples,
in terms of total number of spores collected from spore trap per day in log values.
3) To investigate latent (symptomless) infection of buds and shoots as a tool to predict
disease risk in orchards.
Latent infection of walnut fruit. Data reported last year showed that fruit inoculations from May
to September resulted in a continuous infection and blighted fruit, suggesting that under favorable
conditions immature fruit can be infected latently and fruit will blight by harvest time. Inoculations
late in the season resulted in significantly higher fruit blight than the early inoculations did (data
not shown).
Shoots from fruit inoculations were evaluated for cankers. Percentage of cankers developed from
the Lasiodiplodia citricola inoculations showed an increase over the course of the season (Fig. 2)
with a strong correlation to the time of inoculation. A similar trend was found for canker length
(Fig. 3) as well, though the correlation was not as good. Cankers from Neofusicoccum parvum
inoculations were not found to be correlated with inoculation date.

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Figure 2. Percentage of Vina shoots with cankers from inoculations of fruit clusters with Lasiodiplodia
citricola or Neofusicoccum parvum at 21 day intervals, beginning May 12, 2015 and ending Sep 15, 2015.
Inoculations dates are shown as Julian dates; the cankers were recorded Mar 22, 2016.

Figure 3. Average length of cankers from Vina shoots with fruit clusters inoculated with Lasiodiplodia
citricola or Neofusicoccum parvum at 21 day intervals, beginning May 12, 2015 and ending Sep 15, 2015.
Inoculation dates are shown as Julian dates; the cankers were recorded Mar 22, 2016.

Quantification of latent infection level of shoots using molecular approach. Diplodia species were
not detected in the walnut shoots in any of the seasonal collected samples (Fig. 4). The incidence
of species of Phomopsis in newly-emerged shoots was much lower than those of old shoots (one-
year old) (Fig. 1), indicating very low frequency of latent infection by this pathogen in new shoots
compared with the old shoots. Shoots with latent infections by B. dothidea, Lasiodiplodia spp.
Cytospora spp. and Neofusicoccum spp. were all detected with various incidences (Fig. 4).

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However in comparison, the incidences of latent infection of shoots by Cytospora spp. seemed
lower than those of other three species in July and October (Fig. 4).

1.00 1.00
Phomopsis spp. 0.80
Botryosphaeria dothidea
0.80
0.60 0.60
Incidence of lantent infection of shoots

0.40 0.40
0.20 0.20
0.00 0.00
April July October April July October

1.00 1.00

0.80 Lasiodiplodia spp. 0.80 Cytospora spp.

0.60 0.60

0.40 0.40

0.20 0.20

0.00 0.00
April July October April July October

1.00 1.00
Neofusicoccum spp. Diplodia spp.
0.80 0.80
0.60 0.60
0.40 0.40
0.20 0.20
0.00 0.00
April July October April July October
New shoot One-year shoot New shoot One-year shoot

Month of 2016

Figure 4. Incidences of latent infections of newly-emerged shoots and old, 1-year shoots caused by six
canker-pathogen groups in samples collected in April, July, and October from a commercial walnut orchard
in Butte County. The real-time PCR assay was applied to process these shoot samples.

Comparison in Molecular Severity (MS) between new and old shoots demonstrated that the MSs
were significant higher in old shoots than in new shoots in the samples collected in April for all
the pathogens (Fig. 5). While there was no significant difference in MS between the new and old
shoots for most samples in the later (July and October) samplings (Fig. 5). This trend was still
maintained in the second (in July) sampling for Cytospora spp. and Neofusicoccum spp., while
variations in comparison in MS between newly-emerged and old shoots were found for the other
pathogen groups and in other sampling periods (Fig. 5). The results indicated the possibility of
pathogen movement from old to new shoots during the growing season and some endophytic
features of pathogen species in walnut shoots that need further investigation.

California Walnut Board 11 Walnut Research Reports 2016

The results from latent infection of shoots were quite consistent to those of inoculum quantification
in the air in Objective 2, in terms of component and dominance of the pathogen population in
walnut orchard.

10 10
Phomopsis spp. Botryosphaeria dothidea
8 a 8
a
a a b
Molecular severity of lantent infection of shoots

6 6 a
b b a a a b
4 4
2 2
0 0
April July October April July October

10 10
a Lasiodiplodia spp. Cytospora spp.
8 8
a a a a a
6 b 6 b a a
4 4 b b
2 2
0 0
April July October April July October

10 10
a Neofusicoccum spp. Diplodia spp.
8 a 8
b a a
6 b 6
4 4
2 2

0 0
April July October April July October
New shoot One-year shoot New shoot One-year shoot

Month of 2016

Figure 5. Comparison of mean molecular severities (MS) between newly-emerged shoots and 1-year-old
walnut shoots quantified for each of the six canker-causing pathogens. Three samplings were conducted in
a commercial walnut orchard in Butte County. The real-time PCR was applied to obtain MS data and 32
shoots were processed for each sampling.
Quantification of latent infection level of fruit using the molecular approach. Again, Diplodia spp.
were not detected in fruit in either walnut orchards in Butte Co. or the one at Kearney (KARE)
(Fig. 6). The incidence of fruit with latent infection by Phomopsis was lower than those of other
four pathogens in the Butte Co. orchard (Fig. 6A). In this orchard, the incidence of fruit with latent
infection by B. dothidea was the highest, while those with Lasiodiplodia spp. Cytospora spp. and
Neofusicoccum spp. were quite close (Fig. 6A). The MS values of all the five pathogen groups
were very close to each other with similar standard errors (Fig. 6A).
In the walnut orchard at Kearney, no infections by Phomopsis spp. were detected in fruit (Fig. 6B).
The incidences of the other four canker pathogen groups were quite similar (Fig. 6B), indicating
that B. dothidea, Lasiodiplodia spp. Cytospora spp. and Neofusicoccum were all pathogens which
could cause infections of fruit early in the season. However, no disease by Cytospora on walnut
fruit has been reported.

California Walnut Board 12 Walnut Research Reports 2016

 0.60 8.00

A Buttte Co. Incidence 7.00

0.50
MS
6.00
0.40
5.00

Molecular severity (MS) of fruit latent infection

0.30 4.00

3.00
Incidence of latent infection of fruit

0.20
2.00
0.10
1.00

0.00 0.00
Phomopsis Botryospaeria Lasiodiplodia Cytospora Neofusicoccom Diplodia
spp. dothides spp.
spp. spp. spp.
spp. spp.

0.60 8.00
Incidence
B KARE 7.00
0.50 MS
6.00
0.40
5.00

0.30 4.00

3.00
0.20
2.00
0.10
1.00

0.00 0.00
Phomopsis Botryospaeria Lasiodiplodia Cytospora Neofusicoccom Diplodia
dothides spp. spp.
spp. spp. spp. spp. spp.

Canker-causing pathogen

Figure 6. Incidences of latent infection of walnut fruit and the corresponding molecular severities caused
by six canker-causing pathogen groups for two walnut orchards quantified in May 2016. A total of 30 and
24 fruit were randomly collected from walnut orchards in Butte Co. and KARE, respectively. The qPCR
assay was applied in the quantification approach.

4) To determine the susceptibility of pruning wounds to infection by canker fungi.

Figures 7a and 7b summarize the results of periodic Lasiodiplodia citricola inoculations of
different age (1- to 4-years-old) Chandler shoots, pruned in February and October respectively.
Cankers were infected even sixteen weeks after pruning, which is in contrast to other tree species
(i.e. Prunus spp.) whose pruning wounds usually remain susceptible to canker fungi for only two
weeks after pruning. Overall, there were higher percentages of cankers for the February pruning
dates than the fall dates. Figures 8a and 8b present the percentage cankers for each age of shoot
in relation to inoculation date. The first inoculation resulted in a very high percentage of cankers
in all age shoots in both the February and October prunings. The 3- and the 4-year-old shoots
showed high numbers of cankers throughout the season for the February prunings but were more
variable with significant declines in the October pruned group at the later inoculations.

California Walnut Board 13 Walnut Research Reports 2016

After pooling all the data of inoculation dates by age, it is apparent that the 3- and 4-year-old shoots
showed significantly higher percentages of developed cankers than the 1- and 2-year-old shoots
(Figs. 9a and 9b). This was seen with both February and October prunings. Canker length data
(not shown) exhibited a similar pattern as well. The long period of susceptibility of pruning
wounds of walnut could be partially explained by the fact that there is a central channel in each
shoot filled with loose pith tissues. Apparently, when spraying spore inoculum on the pruning
wounds, fungal inoculum lands in this channel, is absorbed by the pith tissues, and therefore may
survive long in the pith channel, initiating infection internally. Moreover, in the field, one could
easily see that the Botryosphaeriaceae fungi move in advance of the canker (killed tissues) margins
growing in the pith; in some cases, this advanced growth in the pith tissues is more than 1 inch in
length. Thus, to be safe, we suggest that when a grower tries to remove a cankered shoot by
pruning, the cut should be done at least 2 inches below the characteristic margin of canker
externally. Similar recommendations are given when growers prune cankered shoots of pistachio
to manage the Botryosphaeria panicle and shoot blight disease.

California Walnut Board 14 Walnut Research Reports 2016

 a
b 100 a 0 day b
100 Control ab a
80 ab 80
60 a 60
40 40
20 20
0 0

b b 1 week b b
100 3 days
ab 100 ab
80 a 80 a
60 60
40 40
20 20
0 0
Cankers in inoculated shoots (%)

2 weeks a a a b
100 a 100 3 weeks ab
80 ab
80
a
60 60
40 40
20 20
0 0

4 weeks a 8 weeks a a a
100 a a a 100 a
80 80
60 60
40 40
20 20
0 0

b b a a 16 weeks a
100 12 weeks 100 a
80 80
a
60 60
40 a 40
20 20
0 0
1 year 2 years 3 years 4 years 1 year 2 years 3 years 4 years

Age of pruned shoots

Figure 7a. Winter pruning and infection: Percentage of shoots with cankers in 1- to 4-years-old shoots
inoculated periodically with Lasiodiplodia citricola after pruning. Shoots of cultivar Chandler were pruned
on 9-10 Feb 2015 and inoculated from 0 days to 16 weeks after pruning, respectively. Cankers were
evaluated during 4-7 Mar 2016. (There were 10 shoots per age per date of inoculation.)

California Walnut Board 15 Walnut Research Reports 2016

 100.0
Control c 100.0 0 a a a
80.0 bc a
80.0
60.0
60.0
40.0 ab
a 40.0
20.0
20.0
0.0 0.0

c 100.0
100.0 3 days 1 week
80.0 b b
80.0 bc 60.0
60.0
40.0 ab 40.0 ab
20.0 20.0
a
Cankers in inoculated shoots (%)

0.0 0.0

100.0
100.0 2 weeks 3 weeks
b 80.0 b b
80.0
60.0
60.0
ab 40.0
40.0
20.0 a a 20.0 a
a
0.0 0.0

100.0 100.0
80.0 4 weeks 80.0 8 weeks
60.0 a a 60.0
a a a
a
40.0 a 40.0
a
20.0 20.0
0.0 0.0

100.0 100.0 b
b 16 weeks
80.0 12 weeks 80.0
60.0 60.0
a a a
40.0 40.0
a a
20.0 20.0 a
0.0 0.0
1 year 2 years 3 years 4 years 1 year 2 years 3 years 4 years

Age of pruned shoots

Figure 7b. Fall pruning and infection: Percentage of shoots with cankers in 1- to 4-years-old shoots
inoculated periodically with Lasiodiplodia citricola after pruning. Shoots of the cultivar Chandler were
pruned on 27-28 Oct 2015 and inoculated from 0 days to 16 weeks after pruning, respectively. Cankers
were evaluated on 14 Nov 14 2016.

California Walnut Board 16 Walnut Research Reports 2016

 1 year 2 years b b
c
100 100 b
bc bc bc bc ab
ab ab
80 80
bc ab ab a
60 bc 60

Cankers in inoculated shoots (%)

ab

40 a 40

20 20

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120

3 years 4 years
b a a a a
100 a 100 a
b b b b b b b a a
a a
80 80

60 60

40 40

20 20

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120

Inoculation--days after pruning

Figure 8a. Winter pruning and infection: Percentage of Chandler walnut shoots that developed cankers
after inoculation with Lasiodiplodia citricola from 0 to 112 days after pruning. Shoots of ages 1- to 4-years-
old were used in these inoculations; pruning was done on 9-10 Feb 2015; evaluation done during 4-7 Mar
2016.

100 b 1 year 100 c 2 years

80 80

60 60
Cankers in inoculated shoots (%)

a
b
a b b
40 a 40
a
a a
a 20 ab
20 a ab
a
a a
0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120

b 4 years
100 b
100 b
3 years b

c ab
80 80
bc bc ab

a-c ab
60 60
a-c a a
a-c
40 ab 40
a-c
a
20 20

0
0
0 20 40 60 80 100 120
0 20 40 60 80 100 120

Inoculation--days after pruning

Figure 8b. Fall pruning and infection: Percentage of Chandler walnut shoots that developed cankers after
inoculation with Lasiodiplodia citricola from 0 to 112 days after pruning. Shoots of ages 1- to 4-years-old
were used in these inoculations; pruning was done during 27-28 Oct 2015; and evaluation was done on 14
Nov 2016.

California Walnut Board 17 Walnut Research Reports 2016

Figure 9a. Winter pruning and infection: Percentage of pruned 1- to 4-years-old Chandler walnut shoots
that developed cankers after inoculation with Lasiodiplodia citricola spores. The average value for each
year was from 10 inoculation dates after the 9-10 Feb 2015 pruning; measurements were taken during 4-7
Mar 2016.

Figure 9b. Fall pruning and infection: Percentage of pruned 1- to 4-years-old Chandler walnut shoots that
developed cankers after inoculation with Lasiodiplodia citricola spores. The average value for each year
was from 10 inoculation dates after the 27-28 Oct 2015 pruning; measurements were taken on 14 Nov 14
2016.

California Walnut Board 18 Walnut Research Reports 2016

5) To determine efficacy and best timing of fungicide sprays to control Botryosphaeria and
anthracnose blights in walnut.
Protecting pruning wounds. In the February, 2015 in the pruning experiment with Lasiodiplodia
citricola and Neofusicoccum parvum inoculations, there was no significant difference in canker
development among shoots of the different ages or for any of the fungicide treatments (Figure 10a,
10b, 11a, 11b). In addition, there was no significant difference in canker length among the
fungicide treatments. The results in 2016 showed no effect of the fungicides used in this
experiment in reducing canker development as compared to the inoculated but untreated pruning
wounds.

a a a a a a a
100 1 year
90 2 year
Cankers in inoculated shoots (%)

3 year
aa aa a a a a aa
80 4 year

70 a
a
60
a
50
40
30
a
20 b

10
0
n
rol

eal
Top

nd
vo

A bou
Cont

tiS
Meri
ris

n+Vi
Quad

vo
Meri

Figure 10a. Winter pruning and infection: Comparison within treatments of percentage of cankers from
pruned shoots of 4 different ages (1- to 4-years-old) inoculated with Lasiodiplodia citricola. Shoots were
pruned and treated with fungicide 12 Feb 2015 and inoculated the following day (13 Feb); cankers were
measured on 7 Mar 2016.

California Walnut Board 19 Walnut Research Reports 2016

 100 1 year 100
2 years
a a a a a a a
80 80
Cankers in inoculated shoots (%)

a
a
60 a 60

40 40

20 20

0 0
Control Abound Merivon Merivon+VitiSeal Quadris Top Control Abound Merivon Merivon+VitiSeal Quadris Top

a 3 years a a a a 4 years a a
100 100
a a a
80 80

60 60

40 40

20 20

0 0
Control Abound Merivon Merivon+VitiSeal Quadris Top Control Abound Merivon Merivon+VitiSeal Quadris Top

Fungicide treatment

Figure 10b. Winter pruning and infection: Comparison of percentage of cankers within each of the 4
different shoot-age categories (1- to 4-years-old) inoculated with Lasiodiplodia citricola for each fungicide
treatment. Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following
day; cankers were measured on 7 Mar 2016.

1 year
a a a aa a a
100 2 year
3 year
90
Cankers in inoculated shoots (%)

4 year
a a aa a aaaa
80 a a
70
a a
60
50
40
30
a
20 b

10
0
n
rol

eal
Top

nd
vo

A bou
Cont

tiS
Meri
ris

n+Vi
Quad

vo
Meri

Figure 11a. Winter pruning and infection: Comparison within treatments of percentage of cankers from
pruned shoots of 4 different shoot-ages categories (1- to 4-years-old) inoculated with Neofusicoccum
parvum. Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following day;
cankers were measured on 7 Mar 2016.

California Walnut Board 20 Walnut Research Reports 2016

 100 1 year 100
2 years
a a a a a a a a
80 80
Cankers in inoculated shoots (%)

a a
60 60

40 40

20 20

0 0
Control Abound Merivon Merivon+VitiSeal Quadris Top Control Abound Merivon Merivon+VitiSeal Quadris Top

a 3 years a a a 4 years a a a
100 100
a a a
80 80

60 60

40 40

20 20

0 0
Control Abound Merivon Merivon+VitiSeal Quadris Top Control Abound Merivon Merivon+VitiSeal Quadris Top

Fungicide treatment

Figure 11b. Winter pruning and infection: Comparison of percentage of cankers within each of 4 different
shoot-age categories (1- to 4-years-old) inoculated with Neofusicoccum parvum for each fungicide
treatment. Shoots were pruned and treated with fungicide on 12 Feb 2015 and inoculated the following
day; cankers were measured on 7 Mar 7 2016.

In the October, 2015 pruning and fungicide experiment with Lasiodiplodia citricola and
Neofusicoccum parvum inoculations, differences were found with some treatments. Only Merivon
was used to treat inoculations. No significant difference in canker development between shoots
of different ages for either control or Merivon treatments was found in the Lasiodiplodia citricola
inoculations (Figure 12a). No differences were seen between control and Merivon in the 1 year
shoots and in the 2 year shoots. However, there was a significantly higher percentage of cankers
in control and Merivon in both the 3 and 4 years shoots (Fig. 12b). The Neofusicoccum parvum
inoculations resulted in differences between the age categories in the control group but not in the
Merivon treated shoots (Fig. 13a). No differences were seen between control and Merivon in
within any of the age categories (Fig. 13b). These results only showed an effect of the Merivon
reducing canker development in inoculations with Lasiodiplodia citricola in October and not in
any of the Neofusicoccum parvum inoculations. Walnut scale is present at a high level in much of
this orchard now and might have interacted or interfered with the effect of pruning and infection
rates for both pathogens.

California Walnut Board 21 Walnut Research Reports 2016

 1 year
a
100 2 year
3 year
90

Cankers in inoculated shoots (%)

4 year
a
80
70 a

60
a
50
40
30
a a
20
10
a a
0

n
rol

vo
Cont

Meri
Figure 12a. Fall pruning and inoculation: Comparison within treatments of percentage of cankers from
pruned shoots of 4 different ages (1- to 4-years-old) inoculated with Lasiodiplodia citricola. Shoots were
pruned and treated with fungicide on 29 Oct 2015 and inoculated the following day; cankers were measured
on 17 Nov 17 2016.

Figure 12b. Fall pruning and inoculation: Comparison of percentage of cankers within each of 4 different
shoot age categories (1- to 4-years-old) inoculated with Lasiodiplodia citricola for treatment and control.
Shoots were pruned and treated with fungicide on 29 Oct and inoculated the following day; cankers were
measured on 17 Nov 2016.

California Walnut Board 22 Walnut Research Reports 2016

 1 year
b
100 2 year
3 year
90

Cankers in inoculated shoots (%)

4 year
ab ab
80
70 a
a a
60
a
50
40
a
30
20
10
0

n
rol

vo
Cont

Meri
Figure 13a. Fall pruning and inoculation: Comparison within treatments of percentage of cankers from
pruned shoots of 4 different ages (1- to 4-years-old) shoots inoculated with Neofusicoccum parvum. Shoots
were pruned and treated with fungicide on 29 Oct 2015 and inoculated the following day; cankers were
measured on 17 Nov 2016.

Figure 13b. Fall pruning and inoculation: Comparison of percentages of cankers within each of 4 different
shoot-age categories (1- to 4-years-old) inoculated with Neofusicoccum parvum for the fungicide treatment
and control. Shoots were pruned and treated with fungicide on 29 Oct and inoculated the following day;
cankers were measured on 17 Nov 2016.

California Walnut Board 23 Walnut Research Reports 2016

Fungicide trial in Butte County - calendar scheduled sprays. Two recordings were made of disease
symptoms: blighted fruit and blighted (and cankered) current-season shoots (Table 2). They will
discussed separately.
Blighted walnuts were recorded on the day of harvest (28 September 2016) after shaking and
before picking up. The untreated control had 38% blighted fruit. Most fungicide treatments
significantly reduced (P=0.05) the incidence of blighted fruit (Table 2). The best treatment was
the experimental IL-54111 at 15 oz with 6.3% blighted fruit (Trt 16). Next was a mix of Ph-D®
and Tebucon® (Trt 14) with 8.5% blighted fruit. Pristine® (Trt 4) with 9.9% blighted fruit was
next in effectiveness. All of the treatments with Merivon at various timings significantly reduced
blighted fruit (Table 3). The best Merivon® treatment was four sprays at bloom, on 5 May, 10
June, and 12 July. Keep in mind that the blighted fruit from these treatments were recorded before
the postharvest sprays were applied on October 6.
Current season blighted shoots were recorded on 6 December 2016. In this trial, about half of the
fungicides significantly reduced (P=0.05) the incidence of Botryosphaeria canker and blight on
current season spurs (Table 2). The untreated control had an average of 11.2% spurs with cankers.
The best treatment was Luna Experience (Trt 8) with 1.7 blighted shoots. Next was Quash® (Trt
12) and IL-54111 (Trt 16) with 2.7 bighted shoots. Next in effectiveness was a mix of
Fontelis+Abound (Trt 1) with 3% blighted shoots; Regalia® (Trt 22) with 3.1% blighted shoots;
Kenja™ (Trt 15) with 3.3% blighted shoots.
Among a number of treatments with different timings with Merivon®, the timings predicated by
the Leaf Wetness Model (LWM) proved most effective with 3.4 blighted shoots (see timings in
Table 3). This is similar to last year’s results when the Merivon® sprays predicated on the LWM
were the most effective than Merivon® sprayed at other timings. Most other timings that included
a spray on 5 May (Trts 2, 3, 4, and 7) had only slightly more blighted shoots, with the exception
of Trt 6. This is probably because these treatments included an application just before a high risk
infection event during 5-7 May (see Fig. 14).
Fungicide trial in Butte County - best-timing spray. The evaluation of blighted fruit of a spray of
different sets of 5 trees each time with Merivon® suggests that the best timing spray was June 12
(11.2% blight) to the middle of the second week in July (14.3% blight) compared to 38.1% for the
unsprayed control (Table 4). The August spray had a slightly higher incidence of blighted fruit
(15.4%). Earlier (including the bloom spray) had higher incidences of blighted fruit. The
postharvest spray was not evaluated since this spray was after the evaluation date.
Evaluation of blighted shoots on 6 December showed a similar, but not identical trend (Table 4).
The August 10 spray resulted in the lowest incidence of blighted shoots (2.4%) compared to 11.2%
for the unsprayed control. The July (3.6%) and June (3.7%) sprays were very similar. Earlier and
later sprays had higher incidences of blighted shoots.

California Walnut Board 24 Walnut Research Reports 2016

Table 1. Trade name, active ingredient, and class of fungicides used in 2016 in trials to control
Botryosphaeria canker and blight of walnut.

Trade name Active ingredient Class of fungicide

(FRAC number)
Abound® Azoxystrobin (22.9%) Strobilurin (11)
®
Badge X2 Copper oxychloride (23.82%) Inorganic copper (M1)
Copper hydroxide (21.49%)
Fontelis™ Penthiopyrad (20%) Carboxamide (7)
IL-54111 Unknown
Inspire Super® Difenoconazole (8.4%) DMI + Anilinopyrimidine
Cyprodinil (24.1%) (3/9)
KenjaTM* Isofetamid Carboxamide (7)
Luna® Experience SC400 Fluopyram (17.6%) + Carboxamide + DMI-Triazole
Tebuconazole (17.6%) (3/7)
Luna® Sensation 500 SC Fluopyram (21.4%) + Carboxamide + Strobilurin
Trifloxystrobin (21.4%) (7/11)
Manzate® ProStik™ Mancozeb (75%) Carbamate (M1/M3)
Merivon® Fluxapyroxad (21.6%) + Carboxamide + Strobilurin
Pyraclostrobin (21.6%) (7/11)

Movento® 240 SC Spirotetramat Group 23 insecticide

Ph-D® Polyoxin D zinc salt (11.3%) Peptidyl pyrimidine

nucleoside (19)
Pristine® Boscalid (25.2%) + Pyraclostrobin Carboxamide + Strobilurin
(12.8%) (7/11)
Quadris Top™ Difenoconazole (11.4%) + DMI-Triazole + Strobilurin
Azoxystrobin (18.2%) (3/11)
Quash® 50 WG Metaconazole (50%) DMI-Triazole (3)
Regalia® Reynoutria sachalinensis (5%) Biofungicide (P5)
R-106506 SC* Pyraziflum Carboxamide (7)
Serenade® Opti Bacillus subtilis
Tebucon® 45 WP Tebuconazole (45%) DMI-Triazole (3)

WFX-16001 Octanoic (caprylic) acid (42%) Fatty acids

Decanoic (capric) acid (28%)
*Not registered currently for use on walnuts.

California Walnut Board 25 Walnut Research Reports 2016

Table 2. Efficacy of fungicides against Botryosphaeria canker and blight in a Chandler walnut orchard in
Butte County in 2016.

Spray dates Blighted Blighted

Trt Treatment Rate May 5-6 June 10 July 12 August 2 fruit current
(fungicide(s)) &9 (%)1 season
shoots2
(%)
1. Fontelis4 + 20 fl oz Fontelis + Fontelis + Fontelis + Fontelis + 11.2 abc3 3.0 ab
Abound 12.0 fl oz Abound Abound Abound Abound
2. Fontelis+ 20 fl oz Fontelis Fontelis + Fontelis + Fontelis + 14.0 abc 3.9 a-e
Tebuconazole 45 8 oz +Tebucon Tebucon Tebucon Tebucon
DF
3. Fontelis+ 20 fl oz Fontelis + Quadris Fontelis + Quadris 15.6 abc 8.6 d-h
Tebuconazole 8 oz Tebucon Tebucon Top
Quadris Top 14 fl oz
4. Pristine 14.5 oz Pristine Pristine Pristine Pristine 9.9 abc 3.7 a-e
5. Merivon 6.5 fl oz Merivon Merivon Merivon Merivon 12.4 abc 5.4 a-h
6. Quadris Top 14 fl oz Quadris Quadris Quadris Quadris 12.4 abc 3.6 a-d
7. Inspire Super 20 fl oz Inspire Inspire Inspire Inspire 17.9 bcd 6.5 b-h
Super Super Super Super
8. Luna Experience 10 fl oz Luna Exp Luna Exp Luna Exp Luna Exp 16.3 abc 1.7 a
9. 10 fl oz Luna Exp Luna Exp Luna Exp Luna Exp 15.7 abc 5.2 a-h
Luna Experience
Movento Movento Movento Movento
Movento 240 SC
9 fl oz
10. 10 fl oz Luna Exp Luna Exp Luna Exp Luna Exp 13.9 abc 3.6 a-e
Luna Experience
Serenade Serenade Serenade Serenade
Serenade Opti
20 oz
11. Badge+ Manzate 4 lbs Badge+ Badge+ Badge+ Badge+ 11.3 abc 4.1 a-f
ProStick 2.4 lbs Manzate Manzate Manzate Manzate
ProStick ProStick ProStick ProStick
12. Quash 3.5 oz Quash Quash Quash Quash 14.9 abc 2.7 ab
13. Ph-D 6.2 oz PH-D 6.2 PH-D 6.2 PH-D 6.2 PH-D 6.2 20.3 cde 6.4 b-h
14. Ph-D 6.2 oz PH-D 6.2 PH-D 6.2 PH-D 6.2 PH-D 6.2 8.5 ab 4.8 a-g
Tebucon 45 4 oz Tebucon Tebucon Tebucon Tebucon
15. Kenja 17 fl oz Kenja Kenja Kenja Kenja 16.4 abc 3.3 a-c
16. IL-54111 15 fl oz IL-54111 IL-54111 IL-54111 IL-54111 6.3 a 2.7 ab
17. IL-54111 17 fl oz IL-54111 IL-54111 IL-54111 IL-54111 11.1 abc 3.6 a-d
18. Pyraziflumid 3.38 fl oz Pyraz Pyraz Pyraz Pyraz 14.9 abc 4.0 a-e
19. Pyraziflumid 5.08 fl oz Pyraz Pyraz Pyraz Pyraz 10.7 abc 6.2 b-h
20. WXF-160015 0.35% WXF- WXF- WXF- WXF- 15.9 abc 9.4 fgh
16001 16001 16001 16001
21. WXF-160015 0.7% WXF- WXF- WXF- WXF- 12.4 abc 6.4 b-h
16001 16001 16001 16001
22. Regalia 4 quarts Regalia Regalia Regalia Regalia 11.4 abc 3.1 a-c
23. Pristine + 14.5 oz Pristine + Pristine + Pristine + Pristine + 13.9 abc 4.8 a-g
Regalia 4 quarts Regalia Regalia Regalia Regalia
24. Control Untreated 38.1 f 11.2 h

California Walnut Board 26 Walnut Research Reports 2016

Table 2 (cont.)
Dormant and Delayed Dormant—Liquid Lime Sulfur sprays:
Treatment Rate Dormant Delayed dormant Blighted fruit Blighted current
Feb. 25 March 10 (%) season shoots2
(%)
25. Liquid Lime 16 gal. LLS 31.2 def 6.8 b-h
Sulfur
26. Liquid Lime 16 gal. LLS 34.4 ef 10.3 g-h
Sulfur
27. Liquid Lime 8 gal. LLS LLS 37.3 f 8.8 e-h
Sulfur
28. Liquid Lime 15 gal. LLS LLS 30.8 def 7.9 c-h
Sulfur
1 After shaking, 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016.
2 100 current season shoots were recorded on 6 December 2016.
3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05. Statistical analysis was
performed on arcsine transformed data. Values presented were back transformed from the means for the arcsine transformed
data.
4 Dyne-Amic at 0.0625% vol./vol. was added.
5
Sprayed at 200 gallons per acre.

Table 3. Efficacy of various timings of Merivon fungicide against Botryosphaeria blight in a Chandler
walnut orchard in Butte Co in 2016.

Bloom May 5 June 10 July 12 Post- Blighted Blighted

harvest fruit1 (%) current season
March 31 shoots2 (%)
Oct. 6
1. Merivon4 21.2 a1,3 4.7 a2,3
2. Merivon Merivon Merivon Merivon 8.3 a 3.5 a
3. Merivon Merivon Merivon 15.5 a 4.4 a
4. Merivon Merivon 16.9 a 6.4 ab
5. Merivon N.D. 4.1 a
6. Merivon Merivon Merivon Merivon 16.7 a 6.5 ab
7. Merivon Merivon Merivon Merivon Merivon 13.4 a 4.7 a
8. Leaf Wetness Merivon on: 26 April, 23 May, and 20 June 12.9 a 3.4 a
Model-LWM 2016

9. Untreated control 38.1 b 11.2 b

1

After shaking, 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016.
2 100 current season shoots were recorded on 6 December 2016.
3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05. Statistical analysis was
performed on arcsine transformed data. Values presented were back transformed from the means for the arcsine transformed
data.
4 Merivon sprayed at 6.5 fl oz/acre with Dyne-Amic at 0.0625% vol./vol.

California Walnut Board 27 Walnut Research Reports 2016

Table 4. Efficacy of timing of single sprays of Merivon against Botryosphaeria blight in a Chandler walnut
orchard in Butte Co in 2016.

Bloom May 12 June 12 July 10 August Post- Blighted Blighted

April 9 10 harvest fruit1 (%) current season
shoots2 (%)
Nov 3
1. Merivon4 21.2 ab3 4.7 a
2. Merivon 22.2 ab 5.1 a
3. Merivon 11.2 a 3.7 a
4. Merivon 14.3 a 3.6 a
5. Merivon 15.4 a 2.4 a
6. Merivon N.D.5 4.1 a
7. Untreated 38.1 b 11.2 b
1 After shaking, 100 walnut fruit for each of 5 replicated trees were recorded on 28 September 2016.
2 100 current season shoots were recorded on 6 December 2016.
3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05. Statistical analysis was
performed on arcsine transformed data. Values presented were back transformed from the means for the arcsine transformed
data.
4 Merivon sprayed at 6.5 fl oz/acre with Dyne-Amic at 0.0625% vol./vol.
5 Not determined since this treatment was done after harvest.

Fungicide trial to control anthracnose in a Serr orchard in San Benito County.

The San Benito plot which had a history of anthracnose in the past was used again for a fungicide
trial to manage anthracnose. Disease incidence on the leaves and fruit in the plot was recorded on
20 July 2016 and reported in Table 5. Disease incidence was moderate on the leaves in 2016.
Marssonina juglandis, the fungus responsible for anthracnose, was consistently isolated from leaf
lesions before recording. So leaf lesions were considered to be anthracnose. However, lesions on
the fruit consistently yielded the bacterium Xanthomonas campestris, which causes walnut blight.
So fruit lesions were considered to be walnut blight. A mixture of Ph-D+Tebucon (Trt 15) was the
best treatment with 0.2% leaves with anthracnose lesions compared to 41% for the unsprayed
control. Other very effective treatments were Pyraziflumid with 0.3% (Trt 12), Merivon with 0.4%
(Trt 2), and Luna Experience+Movento with 0.6% (Trt 5). Because this orchard has also
Botryosphaeria inoculum and blighted spurs by Botryosphaeria and/or Phomopsis which were
observed in the past, in early spring 2017, buds will be collected for the BUDMON assay. This
assay will provide an evaluation of the long effect of the fungicides used in 2016 against
Botryosphaeria/Phomopsis canker and blight diseases. Results from this plot for fungicide sprays
applied in 2015 are shown in Figure 14. BUDMON results are discussed below.

California Walnut Board 28 Walnut Research Reports 2016

Table 5. Fungicides, rates per acre, dates of application, and efficacy against anthracnose of Serr walnuts
(caused by Marssonina juglandis) and walnut blight (caused by Xanthomonas campestris pv. juglandis) in
San Benito County – 2016.
Trt Treatment Rate Spray dates Fruit Leaves
# (fungicides) per with with
acre walnut anthracn
April 5 April 28 June 1 blight ose
lesions1, 2 lesions1
(%) (%)1
1. Pristine3 14.5 oz Pristine Pristine Pristine 22.7 bc3 2.7 ab
2. Merivon 6.5 fl oz Merivon Merivon Merivon 12.0 ab 0.4 ab
3. Quadris Top 14 fl oz Quadris Top Quadris Top Quadris Top 17.8 abc 5.0 b
4. Luna Luna Exp Luna Exp Luna Exp 17.2 abc 1.9 ab
10 fl oz
Experience
5. Luna 10 fl oz Luna Exp Luna Exp Luna Exp 8.9 ab 0.6 ab
Experience
Movento 240 9 fl oz Movento Movento Movento
SC
6. Luna 10 fl oz Luna Exp Luna Exp Luna Exp 5.0 a 1.1 ab
Experience
Serenade 20 oz Serenade Serenade Serenade
Opti
7. Badge+ 4 lbs Badge+ Badge+ Badge+ 6.8 ab 0.6 ab
Manzate 2.4 lbs Manzate Manzate Manzate
ProStick ProStick ProStick ProStick
8. Quash 3.5 oz Quash Quash Quash 12.9 abc 1.1 ab

9. Kenja 17 fl oz Kenja Kenja Kenja 7.0 ab 3.1 ab

10. IL-54111 15 fl oz IL-54111 IL-54111 IL-54111 8.9 ab 1.6 ab
11. IL-54111 17 fl oz IL-54111 IL-54111 IL-54111 14.8 abc 1.0 ab
12. Pyraz 3.38 fl Pyraz Pyraz Pyraz 8.9 ab 0.3 a
oz
13. Pyraz 5.08 fl Pyraz Pyraz Pyraz 11.0 ab 0.4 ab
oz
14. Ph-D 6.2 oz PH-D 6.2 PH-D 6.2 PH-D 6.2 10.6 ab 2.6 ab
15. Ph-D 6.2 oz PH-D 6.2 PH-D 6.2 PH-D 6.2 11.8 ab 0.2 a
Tebucon 45 4 oz Tebucon Tebucon Tebucon
16. Control Untreate 33.1 c 41.0 c
d
1
100 leaves and fruit for each of 3 replicated trees were recorded on 20 July 2016.
2
Xanthomonas camperstris pv. juaglandis, the causal agent of walnut blight, was isolated from 80% of the Serr
walnut fruit.
3 Numbers followed by different letters are significantly different according to the LSD test at P = 0.05. Statistical analysis was
performed on arcsine transformed data. Values presented were back transformed from the means for the arcsine transformed
data.
4
Dyne-Amic at 0.0625% vol./vol. was added.

California Walnut Board 29 Walnut Research Reports 2016

 60

50 (5/5-7, 34 mm)
Leaf wetness, hours
High Risk
40

30

20 Medium Risk
Low Risk (4/22-23, 16 mm)

10 (617-18, 11 mm)
(5/21, 4 mm)
0
5 10 15 20 25
Temperature, °C
Figure 14. Infection events as predicted by the Leaf Wetness Model (LWM) in 2016 for the Chandler
walnut orchard located next to the Sacramento River in Butte County.

Residual activity in 2016 of fungicides sprayed in the spring of 2015. In the San Benito county
Serr walnut fungicide trial for control of anthracnose, Pristine® and Merivon® were the most
effective at reducing the incidence of Botryosphaeria in dormant buds (Fig. 15). Only Luna®
Sensation reduced the incidence of Phomopsis in these buds (Fig. 16).
BUDMON results for the Butte county Chandler walnut fungicide trial showed that a mix of
Fontelis™ + Abound® most effectively reduced Botryosphaeria in buds (Figure 17). Next in
effectiveness were Merivon® or Pristine®. None of the treatments significantly (P=0.05) reduced
the incidence of Phomopsis in buds (Figure 18), but Luna Sensation seemed to reduce the incidence
to a limited degree. Botryosphaeria was rarely recovered in the BUDMON from the Merivon®
sprays predicated by the Leaf Wetness Model (0.08% - Figure18) compared to 30% from the
unsprayed control. This was the most effective of the various timings in the 2015 fungicide trial.
Other very effective Merivon® timings in reducing Botryosphaeria in the BUDMON assay were
sprays in May, June, July (0.6%), Bloom, May, June, July, and postharvest (0.6%); and May, June,
July, and postharvest (1.1%). The postharvest spray alone, or combined with a bloom spray,
reduced Botryosphaeria in the buds. Only a full season spray program of Merivon® significantly
reduced Phomopsis (0.08%) in the BUDMON assay compared to 8% for the unsprayed control
(Figure 20).
The BUDMON from the single sprays of Merivon® (best timing) followed the results of the prior
season fungicide results (Figure 21). The August 10 spray had the lowest percentage of
Botryosphaeria in the BUDMON assay. The other single spray treatments, with the exception of
the bloom treatment, were less effective; but still significantly (P=0.05) reduced Botryosphaeria

California Walnut Board 30 Walnut Research Reports 2016

in the BUDMON assay. None of the treatments resulted in any significant differences in
controlling Phomopsis compared to the unsprayed control (Figure 22).
BUDMON results from a fungicide trial in Colusa County (Figures 23 and 24), and Yuba County
(Figures 25 and 26) had very low levels of Botryosphaeria and Phomopsis in the unsprayed
controls and none of the fungicide sprays significantly reduced levels of these two fungi in the
buds.

Pristine 14.5 oz a Luna Sensation 7.5 oz a

Merivon 6.5 oz ab Merivon 6.5 oz ab
Luna Sensation 7.5 oz bc Pristine 14.5 oz abc
Quash 3.5 oz bc Quash 3.5 oz abcd
Badge 4 lbs + Manzate 2.4 lbs bc Badge 4 lbs + Manzate 2.4 lbs abcd
Quadris Top 14 oz bc K-Phite 3 qts bcde
K-Phite 3 qts bc Control bcde
Luna Experience 10 oz bc Ph-D 6.2 oz cdef
Control c Fontelis 20 oz def
Ph-D 6.2 oz c Luna Experience 10 oz ef
Fontelis 20 oz c Quadris Top 14 oz f

0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70
Buds with Botryosphaeria (%) Buds with Phomopsis (%)

Figure 15. Effect of 2015-applied fungicides in a Serr walnut Figure 16. Effect of 2015-applied fungicides in a Serr walnut
orchard in San Benito Co. on incidence of Botryosphaeria in orchard in San Benito Co. on incidence of Phomopsis in buds
buds collected during the following winter (2016). A total of 50 collected during the following winter (2016). A total of 50 buds
buds per each of 5 trees were collected on 11February 2016 and per each of 5 trees were collected on 11 February 2016 and
processed using the BUDMON technique. processed using the BUDMON technique.

Fontelis 20 oz + Abound 12 oz a
Merivon 6.5 oz ab
Pristine 14.5 oz ab Luna Sensation 7.5 oz a
Font 20 oz+ Teb 8 oz/Quadris Top 14 oz bc Pristine 14.5 oz ab
Fontelis 20 oz+ Tebuconazole 8 oz cd Merivon 6.5 oz ab
Viathon 4 pints cd Badge 4 lbs + Manzate 2.4 lbs abc
Luna Experience 10 oz cd Control a-d
Fontelis 20 oz+ Vintre 32 oz cde Font 20 oz+ Teb 8 oz/Quadris Top 14 oz a-d
Fontelis 20 oz def Indar 6 oz a-d
Quadris Top 14 oz def Fracture 24.4 oz a-e
Badge 4 lbs + Manzate 2.4 lbs def Quash 3.5 oz a-e
Quash 3.5 oz efg Fontelis 20 oz+ Vintre 32 oz b-f
Fracture 24.4 oz efg Ph-d 6.2 oz+ Tebucon 4 oz b-f
Ph-d 6.2 oz+ Tebucon 4 oz efg Fontelis 20 oz + Abound 12 oz c-f
Luna Sensation 7.5 oz efg Ph-D 6.2 oz c-g
Ph-D 6.2 oz fg Fontelis 20 oz+ Tebuconazole 8 oz c-f
Indar 6 oz fg Fontelis 20 oz c-g
Control g Quadris Top 14 oz efg
Luna Experience 10 oz fg
0 5 10 15 20 25 30 35 Viathon 4 pints g

Buds with Botryosphaeria (%) 0 5 10 15 20 25 30 35

Buds with Phomopsis (%)

Figure 17. Effect of 2015-applied fungicide sprays in a Figure 18. Effect of 2015-applied fungicide sprays in a
Chandler walnut orchard in Butte Co. (Chico) on the incidence Chandler walnut orchard in Butte Co. (Chico) on the incidence
of Botryosphaeria in buds collected during the following winter of Phomopsis in buds collected during the following winter
(2016). A total of 50 buds per each of 5 trees were collected on (2016). A total of 50 buds per each of 5 trees were collected on
18 Feb. 2016 and processed using the BUDMON technique. 18 Feb. 2016 and processed using the BUDMON technique.

California Walnut Board 31 Walnut Research Reports 2016

 Leaf Wetness Model a
May, June, July ab
Bloom, May, June, July, Postharvest a
Bloom, May, June, July, Postharvest ab
May, June, July ab
May, June, July, Postharvest ab
Bloom, May, June, July b Bloom, Postharvest ab
Postharvest c Bloom ab
Bloom, Postharvest c Leaf Wetness Model (3 sprays) b
Untreated Control d May, June, July, Postharvest b
Bloom d Bloom, May, June, July b
Untreated Control b
0 10 20 30 40
Postharvest b
Buds with Botryosphaeria (%)
0 2 4 6 8 10
Buds with Phomopsis (%)

Figure 19. Effect of 2015 sprays of Merivon® applied at Figure 20. Effect of 2015 sprays of Merivon® applied at
various timings in a Chandler walnut orchard in Butte Co. various timings in a Chandler walnut orchard in Butte Co.
(Chico) on the incidence of Botryosphaeria in buds collected (Chico) on the incidence of Phomopsis in buds collected during
during the following winter (2016). A total of 50 buds per each the following winter (2016). A total of 50 buds per each of 5
of 5 trees were collected on 18 Feb. 2016 and processed using trees were collected on 18 Feb. 2016 and processed using the
the BUDMON technique. BUDMON technique.

40
c 10
Buds with Botryosphaeria (%)

c a a
Buds with Phomopsis (%)

30
8
a
6
a a
20

b b 4
ab a a
10 ab
a 2

0
0
Ap

Ju

Ju

Au

No

Un
ay

ne

ly
ril

gu

sp
v

Ap

Ju

Ju

Au

No

Un
3
10
9

12

st

ra
12

ay

ne

ly
ril

gu

sp
v
ye
10

3
10
9

12

st

ra
12
d

ye
10

Figure 21. Effect of 2015 single sprays of Merivon® in a Figure 22. Effect of 2015 single sprays of Merivon® in a d
Chandler walnut orchard in Butte Co. (Chico) on the incidence Chandler walnut orchard in Butte Co. (Chico) on the incidence
of Botryosphaeria in buds collected during the following winter of Phomopsis in buds collected during the following winter
(2016). A total of 50 buds per each of 5 trees were collected on (2016). A total of 50 buds per each of 5 trees were collected on
18 Feb. 2016 and processed using the BUDMON technique. 18 Feb. 2016 and processed using the BUDMON technique.

California Walnut Board 32 Walnut Research Reports 2016

 Fontelis+Tebucon+Postharvest a
Merivon+Postharvest a Fontelis+Tebucon+Postharvest a
Quadris Top a Luna Experience+Postharvest a
Quadris Top+Postharvest a Merivon+Postharvest a
Luna Experience+Postharvest a Quadris Top a
Luna Experience a Control a
Fontelis+Tebucon a Fontelis+Tebucon a
Merivon a Quadris Top+Postharvest a
Control a Luna Experience a
Merivon a
0.0 0.2 0.4 0.6 0.8 1.0
Buds with Botryosphaeria (%) 0.0 0.2 0.4 0.6 0.8 1.0
Buds with Phomopsis (%)

Figure 23. Effect of 2015-applied fungicide sprays in a
Chandler walnut orchard in Colusa Co. on the incidence of
Botryosphaeria in buds collected during the following winter
(2016). A total of 50 buds per replicate tree row were collected
on 25 Feb. 2016 and processed using the BUDMON technique.
Figure 24. Effect of 2015-applied fungicide sprays in a
Chandler walnut orchard in Colusa Co. on the incidence of
Phomopsis in buds collected during the following winter (2016).
A total of 50 buds per replicate tree row were collected on 25
Feb. 2016 and processed using the BUDMON technique.

April/May/June/July (0) a
April/May/June/July (0)a
May/June/July a
May/June/July a a
Untreated Control a
April/May/June a
April/May/June a
Untreated Control a a

0.0 0.2 0.4 0.6 0.8 1.0

Buds with Botryosphaeria (%) 0.0 0.2 0.4 0.6 0.8 1.0
Buds with Phomopsis (%)
Figure 25. Effect of various timings of Merivon® in 2015 in a ®
Figure 26. Effect of various timings of Merivon in 2015 in a
walnut orchard in Yuba Co. on the incidence of Botryosphaeria walnut orchard in Yuba Co. on the incidence of Phomopsis in
in buds collected during the following winter (2016). A total of buds collected during the following winter (2016). A total of 50
50 buds per replicate tree row were collected on 25 Feb. 2016 buds per replicate tree row were collected on 25 Feb. 2016 and
and processed using the BUDMON technique. processed using the BUDMON technique.

LITERATURE CITED

Chen, S.-F., Hasey, J. K., Anderson, K., and Michailides, T. J. 2014. Phylogeny, morphology, distribution,
and pathogenicity of Botryosphaeriaceae and Diaporthaceae from English walnut in California. Plant
Disease 98:636-652.

Luo, Y., Gu, S., Felts, D., Puckett, R. D., Morgan, D. P. and Michailides, T. J. 2017. Development of qPCR
systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops. Journal of
Applied Microbiology (in press).
Morgan, D. P. Driever, G.P. F., Felts, D., Krueger, W. H., and T. J. Michailides. 2010. Evaluation of two
disease systems for Botryosphaeria panicle and shoot blight of California pistachio and efficient control
based on early-season sprays. Plant Disease 93:1175-1181.

California Walnut Board 33 Walnut Research Reports 2016

California Walnut Board 34 Walnut Research Reports 2016