pistachio_anthracnose: Characterization of _Colletotrichum karstii_ and...
In walmes/RDASC: Reproducible Data Analysis of Cientific Cooperations

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The object pistachio_anthracnose includes 9 tables that represent different experiments performed to characterize the pathogen morphology, physiology and pathogenicity.

1	pistachio_anthracnose

The object pistachio_anthracnose is a list containing data.frames. Bellow each data.frame is documented.

The pistachio_anthracnose[["ogrotem"]] stands for optimal growth temperature. This data.frame contains 2646 observations and 8 variables (columns). In this experiment, 7 isolates were cultures onto acidified PDA (APDA) where mycelial growth was daily measured during 7 sucessive days. Mycelial plug of 4 mm was used. Experiment was performed 3 times.

Objectives: (1) Determine the optimum growth temperature per isolate and compare their differences. (2) Compare the isolate AUMGC for each temperature separately.

exp: Integer variable for experiment. This study was performed 3 times.
spp: Character variable for species. In total, two species of Colletotrichum were used: Colletotrichum fioriniae (Cf) and Colletotrichum karstii (Ck).
iso: Character variable for isolate. In total, seven isolates were used, Ck (n = 1, 3G23) and Cf (n = 6, 11J23, 11K11, 11K17, 12D46, 12J05 and 12J41).
rep: Integer variable for repetition. In total, three repetitions or experimental unit (petri plate) were used for each combination of isolate and temperature.
tem: Integer variable for temperature. In total, six temperatures were used to evaluate the mycelial growth of each isolate. Temperatures were: 10, 15, 20, 25, 30, 35^{o}C.
day: Integer variable for day. In total, daily measurements were made during seven sucessive days.
mm1l: Numeric variable for colony diameter 1 (mm). The measurement 1 was taken from one perpendicular colony diameters and recorded in mm. Mycelial plug of 4mm was used as inoculum.
mm2l: Numeric variable for colony diameter 2 (mm). The measurement 1 was taken from one perpendicular colony diameters (perpendicular to mm1) and recorded in mm. Mycelial plug of 4mm was used as inoculum.

The pistachio_anthracnose[["ogertem"]] stands for optimal germination temperature. This data.frame contains 756 observations and 8 variables (columns). The percentual of germinated conidia of Colletotrichum karstii (Ck, n = 1) and C. fioriniae (Cf, n = 6) was evaluated after 6 and 12 hours from incubation, made at six different temperatures. For each combination of isolate (n = 6) and temperature (n = 6), 50 μl of conidial suspension at concentration of 10^{5} was transferred into three water agar plates (2% WA). Six and twelve hours after assay preparation the number of germinated conidia was counted out of 50 conidias. Experiment was performed three times.

Objectives: (1) Determine the optimum germination temperature after 12 hours per isolate and their statistical differences. (2) Compare the isolate frequency of germination for each evaluation (6 and 12 hours) and temperature separately.

exp: As previously described.
spp: As previously described.
tim: Integer variable for time. Two evaluations were made for the same petri plates, the first after six incubation hours and the second after 12 incubation hours at different temperatures.
iso: As previously described.
tem: As previously described.
rep: As previously described.
gerl: Numeric variable germinated conidia. The conidia is considered germinated when its germinative tube is equal or greater than the conidia size. Missing values are reported as NA.
of: Numeric variable for the total number conidia counted. To determine the frequency of germinated conidia, 50 individual conidias were counted/accessed.

The pistachio_anthracnose[["ospotem"]] stands for optimal sporulation temperature. This data.frame contains 189 observations and 8 variables (columns). Following the mycelial growth assay (ogrotem) isolates cultured at 20, 25 and 30^{o}C had their conidia harvested. Each plate (reps = 3 per iso and tem) had 5 or 10 (it depends each case) 5mm mycelial plugs removed from the colony edge. Mycelial plugs were placed inside eppendorf tubes with 1 ml of water and vortexed to release the conidia. Conidia were counted by using an Neubauer Hematocitometer. The experiment was performed three times.

Objectives: (1) Determine the optimum sporulation temperature per isolate and their statistical differences. (2) Compare the isolate sporulation for each temperature separately.

exp: As previously described.
spp: As previously described.
tem: Integer variable for temperature. In this experiment three temperatures were used: 20, 25, and 30^{o}C.
iso: As previously described.
rep: As previously described.
spo: Numeric variable for sporulation. The value represent the total number of conidia counted.
index: Numeric variable used to calculate the conidia concentration according the hematocitometer chambers used. For instance: when I count conidia from the "a" chamber the index used was 1.6x10^{5}. When I count conidia from the "A" the index used was 1x10^{4}. The "A" chamber is located at the four corners of the hematocitometer slide, where one "A" chamber correspond to 16 "a". The "a" = 1/16, then "A" = 0.0625 square-mm. When using the "a" to count conidia I have counted 16 "a" compartments. When using "A" to count conidia I counted 64 "a".
slice: Numeric variable for the mycelial plug area. The equation used to determine the plug area was: A = π r^{2}, where A = area, π = 3.1416, r = plug radius 0.25cm. The plug area was multiplied by the number of plugs used, 10 or 5 plugs.
nsq: Numeric variable for the number of squares. The number of squares "a" and "A" counted. The "a" above mentioned corresponde to 16 squares while "A" corresponde to 64 "a".

The pistachio_anthracnose[["cs"]] stands for conidia size. This data.frame contains 370 observations and 6 variables (columns). Isolates were cultured in APDA for 7 days. After that, conidia was harvested and taken to a microscopy attached to a camera. Pictures were taken and conidia size lenght (len) by width (wid) were measured for 25 single isolates per isolate:experiment by using the software piximetre v 5.2. Experiment was performed two times.

Objectives: (1) Compare the lenght, width and volume of different isolates.

exp: As previously described.
spp: As previously described.
iso: As previously described.
len: Numeric variable for conidia lenght. longitudinal size of conidia measured in micrometre (μm).
wid: Numeric variable for conidia width. Transversal size of conidia measured in micrometre (μm).
vol: Numeric variable for conidia volume. To obtain the conidia volume we used the following formula: Vol = π((wid/2)^2)len. The unit would be μm^{3}.

The pistachio_anthracnose[["af"]] stands for appressorium formation. This data.frame contains 28 observations and 6 variables (columns). Isolates were cultured in APDA for 7 days. After that, conidia were harvested and adjusted to 10^{5} conidia/ml. From the conidia suspension 10 ul was transferred to the surface of a microscopy cover slide that was placed inside a Petri plate contaning 2% WA poured in both plate sides (lid and bottom). Plates were closed and incubated for 24 hours at 25 ^{o}C prior to count the number of germinated conidia forming the appressorium structure. In total 100 germinated conidia were counted. Evaluation was made on microscopy and experiment was performed two times.

Objectives: (1) Compare the frequency of appressorium formation per isolates.

exp: As previously described.
iso: As previously described.
spp: As previously described.
rep: As previously described.
app: Numeric variable for appressorium.
tot: Numeric variable for total number of conidia counted. For each combination of isolate and repetition 100 conidias were counted.

The pistachio_anthracnose[["tos"]] stands for time of susceptibility. This data.frame contains 660 observations and 10 variables (columns). In this study, periodical pistachio cluster inoculations were made every month by using conidial suspensions of 10^{5} for each pathogen species (n = 2). Clusters were covered with plastic and paper bags overnight to allow better infection process and removed in the following morning. Prior to harvest (September) clusters were harvested and each single nut was evaluated for symptoms of anthracnose. The month corresponding to higher blighted nut frequency is the most susceptible period for pathogen infection. The documentation includes data for 2017 and 2018. In September 2019 we will provide the third year results.

Objectives: (1) Determine the period of higher cultivar susceptibility to Colletotrichum karstii and Colletotrichum fioriniae infection for each crop year separately.

yr: Integer variable for year. The year where this experiment was performed.
mo: Integer variable for month. The month correspond to the period of the year that inoculation was made. In 2017, we have three periods of inoculations (June, July and August), while in 2018 we have five periods of inoculation (April, May, June, July and August).
cv: Factor variable for cultivar. In 2017 we inoculated Kerman and Red Aleppo cultivars (n = 2) and in 2018 only Red Aleppo was inoculated (n = 1).
spp: As previously described.
fla: Factor variable for flag. different flag colors were used to identify spp and period of inoculation used. They dont need to be considered on the analises.
arb: Integer variable for tree. Every month, three trees were used per combination of cultivar and specie.
clu: Integer variable cluster. Each combination of mo:cv:spp:arb include 10 pistachio clusters that were inoculated. The cluster can be used as a repetition for each tree.
bli: Numeric variable blighted nuts. Its the number of nuts that were blighted due to the pathogen infection. Missing values are reported as NA.
hea: numeric variable for healthy nuts. Its the number of nuts that were found to be healthy meaning: no symptoms of anthracnose were observed. Missing values are reported as NA.
tot: numeric variable for total number of nuts counted per cluster. The number may vary from cluster to cluster. Missing values are reported as NA.

The pistachio_anthracnose[["pato_vv"]] stands for pathogenicity performed in vivo. This data.frame contains 360 observations and 10 variables (columns). In 2017 and 2018, inoculations were performed as described for (tos). The following data set include the inoculation made on June for each year on different pistachio cultivars. Data for 2019 will be available in September this year.

Objectives: (1) Compare the cultivar susceptibility to Colletotrichum karstii and Colletotrichum fioriniae infection.

yr: As previously described.
mo: Integer variable for month.
cv: Factor variable for cultivar. in 2017 we inoculated Kerman, Golden Hills and Red Aleppo cultivars. In 2018 we inoculated Kerman, Golden Hills and Red Aleppo. In 2019 (data not yet available) we inoculated Kerman, Golden Hills, Lost Hills and Red Aleppo.
spp: As previously described.
fla: As previously described.
arb: As previously described.
clu: As previously described.
bli: As previously described.
hea: As previously described.
tot: As previously described.

The pistachio_anthracnose[["pato_vt"]] stands for pathogenicity in vitro. This data.frame contains 5760 observations and 10 variables (columns). The study was performed on detached leaves of Kerman and Red Aleppo cultivars. Each cultivar was inoculated with mycelial plugs (4mm) of Colletotrichum karstii and Colletotrichum fioriniae incubated at 20, 25 and 30 ^{o}C. For each combination of temperature, species and cultivar 30 leaves were prepared. Lesion size was measured at 3, 5, 7 and 10 days after inoculation. The study separate kerman from red aleppo, but randomized species within cultivars. The experiment 1 was the only experiment replication that was not randomized, meaning that each crysper (plastic container) held a single cultivar and specie.

Objectives: (1) Determine the optimum temperature for lesion growth for Colletotrichum karstii and Colletotrichum fioriniae inoculated on Red Aleppo and Kerman separately. (2) Compare the susceptibility of cultivar to each species separately.

exp: As previously described.
tem: As previously described.
cv: As previously described.
cri: Integer variable for crysper. The crysper is the plastic container that holds 10 leaves each.
lea: Integer variable for leaf. The leaf is the experimental unit from where lesion size was measured.
spp: As previously described.
iso: As previously described.
day: As previously described.
mm1: As previously described. Notice that measurement need to be divided by 100 to obtain the correct size in mm. Missing values are reported as NA.
mm2: As previously described.Notice that measurement need to be divided by 100 to obtain the correct size in mm. Missing values are reported as NA.

The pistachio_anthracnose[["spo_vt"]] stands for sporulation in vitro. This data.frame contains 108 observations and 10 variables (columns). Following the pathogenicity study in vitro (above described) 10 lesions caused by Colletotrichum karstii and Colletotrichum fioriniae were detached from the leaves and combined into three new repetitions, according the temperature used. For instance: The 30 Ck lesions obtained at 20^{o}C on Kerman cv originated three reps of 10 lesions combined and placed in different flasks. Water was added to release the spores from the lesions. Sporulation was performed according conventional protocol above described.

Objectives: (1) Determine the optimum sporulation temperature for Colletotrichum karstii and Colletotrichum fioriniae on Red Aleppo and Kerman cultivar. (2) Compare sporulation capacity of Ck and Cf at different temperatures and cultivars.

exp: As previously described.
spp: As previously described.
tem: As previously described.
iso: As previously described.
rep: As previously described.
cv: As previously described.
spo: As previously described.
index: As previously described.
slice: As previously described. Sum of lesion size combined (n = 10)
ml: Integer variable for water volume used to harvest conidia from leaf lesion.