05/02/2018
1
“Sentinel nursery
monitoring for pathogens: the
example of the Fuyang plot” Andrea Vannini & Carmen Morales Rodriguez
This presentation aims to provide
qualitative and quantitative elements to
evaluate the work needed in the inspection of a nursery/plantation, and
that might be useful for the
standardization of methodology
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Where and when
The ‘sentinel nursery’ was established in 2012 in the district of Fuyang in the province of Zhejiang, in the frame of the EU project ISEFOR
The site was characterized by bamboo tropical forest; tea plantations and additional horticulture crops
Climate was warm and humid (34-36°C)
Five chinese ornamental tree species among the most traded to Europe: Fraxinus chinensis; Acer palmatum; Buxus microphylla; Ilex cornuta; Zelkova schneideriana
Five blocks with 20 individuals (sub-block) per each species; a total of 100 individuals per species; 500 trees in total
Estimated age 6-8 years
Where and when
The ‘sentinel nursery’ was established in 2012 in the district of Fuyang in the province of Zhejiang, in the frame of the EU project ISEFOR
The site was characterized by bamboo tropical forest; tea plantations and additional horticulture crops
Climate was warm and humid (34-36°C)
Five chinese ornamental tree species among the most traded to Europe: Fraxinus chinensis; Acer palmatum; Buxus microphylla; Ilex cornuta; Zelkova schneideriana
Five blocks with 20 individuals (sub-block) per each species; a total of 100 individuals per species; 500 trees in total
Estimated age 6-8 years
05/02/2018
3
Where and when
The ‘sentinel nursery’ was established in 2012 in the district of Fuyang in the province of Zhejiang, in the frame of the EU project ISEFOR
The site was characterized by bamboo tropical forest; tea plantations and additional horticulture crops
Climate was warm and humid (34-36°C)
Five chinese ornamental tree species among the most traded to Europe: Fraxinus chinensis; Acer palmatum; Buxus microphylla; Ilex cornuta; Zelkova schneideriana
Five blocks with 20 individuals (sub-block) per each species; a total of 100 individuals per species; 500 trees in total
Estimated age 6-8 years
Where and when
The ‘sentinel nursery’ was established in 2012 in the district of Fuyang in the province of Zhejiang, in the frame of the EU project ISEFOR
The site was characterized by bamboo tropical forest; tea plantations and additional horticulture crops
Climate was warm and humid (34-36°C)
Five chinese ornamental tree species among the most traded to Europe: Fraxinus chinensis; Acer palmatum; Buxus microphylla; Ilex cornuta; Zelkova schneideriana
Five blocks with 20 individuals (sub-block) per each species; a total of 100 individuals per species; 500 trees in total
Estimated age 6-8 years
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Where and when
The work has been carried out in
the period August 5-September 3,
2017
Scientists involved: Carmen
Morales Rodriguez & Andrea
Vannini from Europe; Dr Yongiou
Wang and his staff from China
Samples processing has been
carried out at the lab of Dr.
Yongiou Wang at the School of
Forestry and Biotechnology of the
Zhejiang A&F University in Lin’an
Where and when
The work has been carried out in
the period August 5-September 3,
2017
Scientists involved: Carmen
Morales Rodriguez & Andrea
Vannini from Europe; Dr Yongiou
Wang and his staff from China
Samples processing has been
carried out at the lab of Dr.
Yongiou Wang at the School of
Forestry and Biotechnology of the
Zhejiang A&F University in Lin’an
05/02/2018
5
Preliminary activities before departure
Exchange of mails with Dr. Wang in order to:
Inform and share the objectives of the visit and the methodology to be applied (about 45 days in advance)
Agree on the time-sheet of the proposed activities and test of feasibility (about 1 month in advance)
Determine the equipments needed versus equipments available at the laboratory in China (about 1 month in advance)
Determine the consumables needed versus consumables available (about 1 month in advance)
Fill in a list of materials and small equipment to ship to China (about 20 days in advance)
Purchase of needed materials and equipment (in our case DNA extraction kits from plants and soil; culture media)
Scheduled activities in China: biological diagnosis
Sampling of symptomatic plant tissues for determination of fungal associated community
Symptoms classification
Collection of soil samples for determination of soilborne oomycetes community
Isolation of fungal community from symptomatic tissues onto generic media (PDAsa)
Baiting of soil samples for oomycetes community enrichment
Isolation of oomycetes community on selective media (PARP)
Grouping of pure fungal and oomycetes colonies per morphotype
Growth of morphotyes onto solid (fungi) and liquid (oomycetes) media for DNA extraction (single hypha isolation)
DNA extraction from colonies
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Scheduled activities in China: NGS analysis
Sampling of no-symptomatic
tissues from each species: leaves
and woody tissues
Lyophilization of samples
DNA extraction
Sampling 1
Sampling has been carried out in 2 rounds (different days)
First round: samples from symptomatic trees were collected
At a first instance tissues and soil from each individual were kept separated, but this was increasing to much the number of samples
Thus the sub-block (individuals of the same species within a block) was considered as unit of sampling for both symptomatic tissues and soil
Each sample was stored in a separate bag tagged per species and block and kept in a cold-box
Back to the lab, the samples were stored in a cold room until the following day when processing started
The media was prepared for next days isolation
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Symptoms classification
Up to 18 different symptoms were
assessed on the 5 host species
A conservative approach was
used for symptoms classification giving different codes to similar
symptoms.
Symptoms code included a
number and the host species.
Samples processing
Samples from symptomatic tissues were processed as follow:
Small fragments were obtained (1 square cm max) at the interface of healthy and necrotic tissues
Two different sterilization methods were employed and compared: i) 30 s sterilization in 75% EtOH followed by 3 washings in sdH2O; ii) 1 min in 75% EtOH; 3 min in 2% sodium hypochloride; 30 s in 75% EtOH; 3 washings in sdH2O.
After sterilization the fragments were cutted in smaller pieces and plated onto PDAsa.
A total of 600 fragments were plated
Plates were incubated at 20°C
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Samples processing
Soil samples were processed as follow:
About 200 gr of soil from each sample was put in a tray and covered with sdH2O
Debris floating on the surface were removed and different baits were put floating on the water surface
During the following 6 days, baits showing necroses were removed, cut in smaller fragments, sterilized in 75% EtOH for 30s, washed 3 times in sdH2O and plated on selective media for oomycetes (PARP)
Plates were incubated at 20°C
Samples processing
Soil samples were processed as follow:
About 200 gr of soil from each sample was put in a tray and covered with sdH2O
Debris floating on the surface were removed and different baits were put floating on the water surface
During the following 6 days, baits showing necroses were removed, cut in smaller fragments, sterilized in 75% EtOH for 30s, washed 3 times in sdH2O and plated on selective media for oomycetes (PARP)
Plates were incubated at 20°C
05/02/2018
9
Samples processing
Soil samples were processed as follow:
About 200 gr of soil from each sample was put in a tray and covered with sdH2O
Debris floating on the surface were removed and different baits were put floating on the water surface
During the following 6 days, baits showing necroses were removed, cut in smaller fragments, sterilized in 75% EtOH for 30s, washed 3 times in sdH2O and plated on selective media for oomycetes (PARP)
Plates were incubated at 20°C
Morphotypes selection & DNA extraction
Fungal colonies
Fungal biodiversity from tissues was very high regardless the sterilization method applied (this was expected dealing with a tropical environment)
Up to 10 different morphotypes per symptom were obtained
Up to 2 successive transfers of mycelial fragments onto fresh PDAsa were necessary in order to obtain pure cultures
180 pure cultures were obtained, resulting in a number of morphotypes estimated conservatively in 79
Morphotypes were duplicated onto PDA and the new set used for DNA extraction using the ‘plate scrape’ method
At least 1 DNA extraction per morphotype was carried out
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Morphotypes selection & DNA extraction
Oomycetes colonies
More than 20 Phytophthora colonies (23) were obtained in pure culture
A total of 6 Phytophthora spp. morphotypes were identified from soil samples
Phytophthora morphotypes were grown in 1,5 ml Eppendorf tubes in liquid CA
More than 1 DNA extraction per morphotype was carried out
Sampling 2
Second round: samples from asymptomatic tissues were collected
Again, sub-block was considered as sampling unit
Each sample was stored in a separate bag, tagged for species and block, and kept in a cold-box
Back to the lab the samples were stored in a cold room until the following day when processing started.
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Sample processing
Asymptomatic plant tissues were
lyophilized for 24-36 hrs at -50°C
under vacuum
Each sample was then grinded in
1,5 ml Eppendorf tube with sea
sand
The powder obtained was used
for DNA extraction
A total 50 samples was processed
(5 species x 5 blocks x 2 types of
tissue)
Data records
All datas were organized in Excel
files
Morphotypes.xlsm
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Missed activities
We had no time to carry out morphological identification of fungi and oomycetes
Very light colony and reproductive structures observations were carried out bringing to the identification at genus level of Fusarium, Pestalotiopsis, Phytophthora
At least, 2 additional weeks would have been necessary to address the morphological identification
For biosecurity reasons, no isolates were took back to Europe
At the moment we are scheduling an additional visit to the lab of Dr. Wang to address these issues
Scheduled activities back to Europe
DNA barcoding of fungal and
oomycetes morphotypes
NGS processing of DNA from no-
symptomatic samples
Data elaboration
Fungi and oomycetes collections
are maintained at the laboratory
of Dr. Wang in Lin’an
05/02/2018
13
Minimal person/month efforts
To carry out the activities abroad a minimum of 3 person/month are needed over a period of 30 days (21 working days)
In the reality, the need is higher since average working time exceeded the 8 hrs per day
In our case, 1 person/month was provided by each Dr Morales and Dr Vannini, while 1 person/month in total was provided by the staff of Dr Wang
Additional 2 persons for 2 weeks (1 person/month) are required If we include additional activities such as morphological identification of specimens,
A minimum of additional 2 person/month are required for DNA barcoding at DIBAF-UNITUS
Total 6 person/month
Required skills
The core team should be represented by a minimum of 2 scientists possibly trained to work together
Required skills are: experience in survey and sampling in natural environments; biological diagnosis; DNA extraction; molecular barcoding; good experience in fungi and oomycetes manipulation and taxonomy; good knowledge of bioinformatics
The best would be having both scientists trained in all the techniques. Complementarity is also OK
Supporting team collegues should also have basic knowledge in biological and molecular diagnosis (we were very lucky in this)
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Logistic
An agreement is needed
between the 2 laboratories
regulating:
Extent of collaboration
Reciprocity
Use of produced data
Mid-term activities to carry out
Funding and costs
Costs of Dr. Morales (travel, accomodation & food) have been covered by the STSM fellowship (2.500 Euro)
Costs of Dr. Vannini (travel, accomodation & food) have been covered by his own saving (2.500 Euro)
Costs of logistic: car and driver to get to the sampling site were covered by both STSM fellowship and Dr. Vannini own savings (about 150 Euro).
Costs of materials have been covered by DIBAF-UNITUS: DNA extraction kits and culture media (2.000 Euro); and Dr. Wang: Petri dishes; chemicals; antibiotics etc. (?)
Foreseen costs to complete analyses covered by DIBAF-UNITUS: sanger sequencing (about 500 euros); NGS sequencing (estimated 1.500-2.000 Euro); woperson power (5.000 euro)
Foreseen costs at Dr. Wang laboratory to keep the maintain the fungi and oomycetes collection (?)
Total costs: exceeding 15.000 Euro
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Efficient use of the STSM instrument for similar activities
Unless an established collaboration with the hosting Institution, a minimum of 2 applicants are needed to assure the achievement of the objectives
Considering a similar experimental design, or a similar number of replicates, in order to complete DNA extraction from isolates and plant tissues, 4 weeks are needed
Hos Institution not necessarily guarantees equipment and materials. This aspect has to be discussed, detailed and agreed before the STSM begins.
According to these considerations we can summarize: 2 applicants x 4 weeks and at least 1 scientist at the host institution taking care of local logistic and supporting the laboratory work
Let’s start from such
experiences to agree on
common protocols
Thanks for your attention
