The quality and quantity of nut production are fundamental to the economic viability of chestnut cultivation, yet recent reports indicate that severe damage due to moulds represents a significant problem for growers. We carried out an investigation of the agents of chestnut rot and internal fruit damage in three orchards in Italy. Black and brown rot, as well as insect damage, were found in all the areas examined. Brown rot appeared to be the main cause of damage, affecting 8% to 49% and 2% to 24% of nuts collected from the ground and from burrs, respectively. With respect to morphology and DNA sequencing analyses, fungal isolates obtained from brown rot were homologous with
Chestnut cultivation has progressed in Italy and other European countries as a result of a favourable market and the recovery of trees following natural establishment of hypovirulence in chestnut blight (
The recent appearance of the invasive Asian chestnut gall wasp (
In this study, we investigated brown rot damage reported in three different Italian chestnut-growing areas in Piedmont, Trentino and Tuscany with the aim of isolating and identifying the causal agents, verifying their homology, comparing their morphology, testing their pathogenicity and formulating hypotheses regarding the factors responsible for their spread.
Chestnut orchards were selected in October 2011 in three Italian regions: Boarda (Val di Susa, Turin, Piedmont), Montesenario (Mugello, Florence, Tuscany) and Crosano (Val d’Adige, Trento, Trentino-South Tyrol). The general environmental features of each stand are reported in
Monthly mean temperatures and accumulated precipitation for 2011 were obtained from the meteorological stations closest to each of the study sites: Castel Borello (630 m a.s.l. - Società Meteorologica Italiana network), Borgo San Lorenzo (193 m a.s.l. - LaMMA network), and Besagno (382 m a.s.l. - FEM agro meteorological network). Only raw, non-validated, non-gap-filled meteorological data series were used. For Besagno, monthly mean temperatures and accumulated precipitation recorded during the vegetative period (April-September) were extracted from data for the whole period 2001-2011. These monthly values were compared with the average of the 10 years data series.
Three chestnut trees were randomly selected in each of the orchards investigated. Forty nuts were collected from burrs still attached to the branches of each tree and sixty nuts were collected from the ground under the same trees. Burrs were examined for perithecia presence. Fruit samples were packaged separately in paper bags and immediately transferred to the laboratory for assays. Each nut was dissected and examined: fruits showing brown rot were separated from nuts affected by black rot and selected for isolation. A further 10 nuts were collected from ground in each area, stored in field conditions and evaluated for the presence of fruiting bodies.
Ten shoots were randomly collected from the crown of each tree, placed in plastic bags and stored at 4 °C for laboratory assay.
Tissue from the initial decaying endosperm and from mummified fruits were surface- sterilized for 1 min in 70% ethanol, 5 min in 1.25% sodium hypochlorite and 30 sec in 70% ethanol. They were then rinsed twice in sterile, distilled water and blotted on dry sterile filter paper. Using a sterile scalpel, fragments of infected tissue were plated onto Potato Dextrose Agar (Difco, USA) containing biotin (1mg/l) and methionine (100 mg/l - PDAmb) and incubated in Petri dishes in the dark at 25 ± 1° C for seven days.
Fungal cultures were sub-cultured onto PDAmb in the dark at 25°C.
Morphological observation of colonies was carried out with an SMZ1000 stereomicroscope (Nikon, USA), while microscopic features, such as size, shape, color and arrangement of conidia, were observed with an Eclipse 80i microscope (Nikon). Measurements of 50 pycnidia and 50 conidia of one strain from each area plus 50 conidia collected from fructifications on nuts were made from digital sight images (DS-L2 imaging controller, Nikon, USA).
Brown rot mycelium was placed in 10 ml of sterilized tap water and shaken. One ml of conidial suspension (2 x 103 conidia ml-1) was diluted 1:5 with sterile tap water and plated on Petri dishes containing PDAmb substrate. Individual germinated conidia were collected under sterile conditions with a micro-needle using a stereomicroscope then transferred to plates and incubated under the conditions reported above. Thirty six individual conidial strains from each area were examined for morphological characteristics.
An 8-10 cm section was cut from each shoot of the current years’ and the two-year growth. Each sample was surface disinfested for 10 sec in 95% ethanol followed by 4 min in 2% NaOCl solution with 2 drops of Tween 80 l-1 (
Isolations were incubated at 25 ± 1 °C for 7 days in the dark, after which the growing fungi were identified by their cultural characteristics. The number of fragments colonized by each type of culture was linked with the original sample and plot.
DNA assays were carried out using cultures from individual monoconidial strains randomly selected from each area, two from Crosano and one from each of the other sites. Genomic DNA was extracted from approximately 50 mg of mycelia scraped from the surface of a 7-10 days culture growing on PDAmb. DNA was also extracted directly from initial decaying, brown tissue and chalky tissues in symptomatic nuts from Crosano. A total of seven strains were so assayed.
DNA was extracted using the NucleoSpin1 Plant Kit according to the manufacturer’s instructions (Macherey-Nagel, Düren, Germany). Internal transcribed spacer regions 1 and 2, including the 5.8S rDNA, were amplified and sequenced using the primers ITS5 and ITS4 (
PCR products were purified using Exo-SAP (Euroclone S.p.A., Italy) following the manufacturer’s instructions. Amplified products were sequenced with the Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 3130xl Genetic Analyzer. BLASTN comparison of the amplicon sequences was carried out using the NCBI database to confirm the identity of the strains. Raw sequences were edited using Sequencer version 4.5 for Windows (Gene Codes Corporation, Ann Arbor, MI, USA) and deposited in the GenBank.
Nucleotide sequences were aligned with other fungal sequences (
Some 105 asymptomatic nuts were collected in a chestnut orchard near Trento (Lisignago), where no brown rot symptoms have been recorded until now. A small sample (15 nuts) was randomly chosen and dissected to confirm healthy status. Sixty of the remaining nuts were surface-sterilized for 1 min in 70% ethanol, 5 min in 1.25% sodium hypochlorite, 30 sec in 70% ethanol and rinsed in sterilized tap water. After this treatment, the fruits were artificially infected with each of the monoconidial strains selected for the DNA assay. An artificial wound 0.5 cm deep was made in the pericarp at the apical part of the fruit using a sterilized scalpel, as reported by
The statistical analysis concerned the symptoms presence and isolations from nuts and shoots. For each symptom detected on nuts, considering the mean value of the three sampled trees, the three sites were compared using the Chi-square (χ2) test for 2-way tables (coupling the sites two by two). The same analysis was used for fungal isolation from bark tissue of shoots. χ2 values were compared with critical χ2 value at significance level α=0.05 and 1 degree of freedom.
The three sites, located at similar altitudes, vary in exposure and morphology (
During 2011, high temperatures (over 20 °C) were recorded for August and September, while in the same months rainfall was less than 100 millimeters at all three stations (
Significant differences were found between healthy fruits collected from the ground at Crosano and those collected at the other two sites (
The main symptoms recorded in the remaining rotten nuts of the samples were of black rot (
Initial and final symptoms coexisted in most of the nuts and, in a few cases, black and brown rot were observed together. Both rots were present in the nuts collected from the ground and in those still on the trees, although the extent of damage was greater in the nuts on the ground: black rot affected 8% of nuts gathered from the ground and 2% of nuts on the trees in the Boarda area, while the figures for Montesenario were 7% and 1%, respectively, and for Crosano 3% and 0%, without significant differences among sites (
Brown rot was widespread in the Crosano area, where 49% of the nuts from the ground and 24% of the nuts from the trees showed symptoms. In this stand, the disease was significantly more present than in the other two sites either in nuts taken from the ground (vs. Boarda: χ2=19.21, p<0.05;
Storage of the nuts, whether controls or producing fruiting bodies in natural conditions, exacerbated the brown rot as all the fruits displayed chalky tissue after three months. An abundance of pycnidia, first brownish-grey then blackish, appeared on the surface of the endosperm below the episperm (
Cultures of greyish-black mycelia were isolated from black rot tissue and identified as the species
Colonies with light brown mycelium were isolated from initial brown tissue and white, chalky tissue. Orange masses of conidial growth appeared in concentric rings on the surface of the cultures (
High percentages of positive isolation were obtained from the bark tissue of both one-year and two-years-old shoots at each site (
Morphological identification of fungal isolates was confirmed with molecular data obtained from DNA amplification. Four ITS sequences obtained from the DNA of fungal cultures and three sequences obtained directly from nut tissue were analyzed. A Megablast search in NCBI revealed the above fungal specimens having the highest similarity with the
Phylogenetic analysis of
All fifteen nuts assayed to test their healthiness were found to be uncontaminated by moulds.
The
A high degree of damage was found in all three orchards sampled and both black rot and brown rot fungi were common, especially in the Crosano orchard where more than half of the crop appeared to be affected by mould fungi. Insect damage was also severe, confirming its destructiveness in nut production.
The unusual spread of brown rot, the high incidence of damage caused by this disease and the extent of the damage found in Crosano justifies growers’ worries about this disease, which has been highly destructive in other parts of Piedmont and Tuscany (
According to the owner of the Crosano orchard, brown rot has been present since 2003, although its presence has increased in the last three years. Its emergence does not appear to be related to the appearance of
Meteorological records report a warm, dry period in all the study areas during the 2011 vegetative season, but in the area least affected (Boarda) irrigation helped the trees. Stress and drought periods have been common in the Crosano area since 2003. Plant stress brought about by drought and gall wasp attacks may have favored brown rot spread in central and northern Italy. Afflicted and suffering chestnut trees are likely to be highly susceptible to infection by endophytes or latent pathogens such as
In conclusion,
We would like to thank Giovanni Falchero, Paolo Miorelli, Luciano Massetti and Francesca Ugolini for technical assistance in the field and data processing.
Monthly precipitation and mean temperatures recorded during 2011 at the three meteorological stations closest to the study sites.
(a) Nut completely destroyed by black rot due to
Dendrogram showing the similarities between amplificates from mycelium isolates and nut tissue samples (boldface) and sequences in the NCBI database created by the CLUSTALW software.
Main environmental features of the study sites.
Site | Boarda | Montesenario | Crosano |
---|---|---|---|
Elevation (m a.s.l.) | 565 | 650 | 550 |
Exposure | North | North West | North East |
Morphology | Terrace | Terrace | Slope |
Slope | 0% | 5% | 15% |
Substrate | Alluvial | Sandstone | Turbidite |
Management | cultivated | Semi-cultivated | Semi-cultivated |
Irrigation | Twice (July, August) | absent | absent |
Geographic coordinates | 45.120369 N7.187719 E | 43.891551 N11.330627 E | 45.827764 N10.975599 E |
Percentages of damaged nuts found in the samples collected from the three different chestnut orchards. Chi-squared test 2-way tables was performed. Critical χ2 value was at α=0.05 and df = 1. (*): values which differ in a significant way.
Symptoms | Nuts from the ground | Nuts from burrs | ||||
---|---|---|---|---|---|---|
Boarda | Montesenario | Crosano | Boarda | Montesenario | Crosano | |
No Symptoms (%) | 62 | 65 | 32 | 81 | 72 | 56 |
Black rot (%) | 8 | 7 | 3 | 2 | 1 | 0 |
Brown rot (%) | 11 | 8 | 49* | 2 | 4 | 24* |
Insect damage (%) | 19 | 20 | 16 | 15 | 23 | 20 |
Comparison of conidiomata, conidia and cultural characteristics of fungi associated with brown rot obtained in this study compared with other findings reported in the literature. All studies cited reported the fungus on
Strains | Perithecia(μm) | Conidiomata(μm) | Conidia(μm) | Cultural morphology | Symptoms |
---|---|---|---|---|---|
- | 200 x 158 | 5.1-8.9 x1.9-3.9 | Light brown mycelium. Orange masses of conidiomata in concentric rings | Brown rot, endophytic on shoots | |
n.r. | n.r. | 6-8 x 2-3 | Not reported | Brown rot | |
- | 200 x 160 | 6-7.5 x 2.4 | Light brown mycelium. Orange masses of conidiomata in concentric rings | Brown rot | |
- | n.r. | 8.1 x 2.4 | White mycelium in concentric rings | Brown rot, necrosis on shoots and leaves | |
- | n.r. | 5.5-8.0 x2.0-3.0 | Light brown mycelium. Orange conidiomata in concentric rings | Colonisation of |
|
- | 244-268 x146-190 | 4.9-7.3 x2.4-3.7 | Greyish brown mycelium with concentric rings of conidial stroma | Brown rot, endophytic on shoots | |
238-242 | - | 6.5-9.5 x2.0-4.0 | Greyish brown mycelium with concentric rings of conidial stroma | Brown rot, endophytic on shoots |
Mean percentage of fungi isolations from bark tissue of healthy shoots (one- and two-years old) of chestnut plants. The Chi-squared test for 2 way table was performed (presence/no presence for each isolate and coupled sites). Critical χ2 value at α=0.05 and df=1. (*): values which differ in a significant way.
Shoots | Species | Crosano | Montesenario | Boarda |
---|---|---|---|---|
1 year old | 70* | 33 | 37 | |
|
0 | 1 | 0 | |
other fungi | 21* | 4 | 9 | |
Sterile fragments | 9 | 61 | 55 | |
2 years old | 71* | 48* | 33 | |
|
4 | 1 | 3 | |
other fungi | 17* | 4 | 9 | |
Sterile fragments | 9 | 47 | 55 |
Database matches for sequences generated from ITS 5 and ITS 4 PCR products, derived from tissue showing symptoms of brown rot and mycelia of monoconidial cultures.
Isolate | DNA origin | Description | Max ident.(%) |
---|---|---|---|
Gm_TN1 | Trentino mycelium | 99 | |
Gm_TO | Piedmont mycelium | 100 | |
Gm_FI | Tuscany mycelium | 99 | |
Gm_TN2 | Trentino mycelium | 99 | |
Gn_TN3 | Chalky tissue | 99 | |
Gn_TN4 | Brown tissue | 99 | |
Gn_TN5 | Initial brown tissue | 99 |
Results of the artificial inoculation tests. (*): 13% (2 nuts) affected by
Strain | % nuts affected by brown rot | % asymptomatic |
---|---|---|
Gm_TN1 | 71 | 29 |
Gm_TN2 | 72 | 28 |
Gm_FI | 62 | 38 |
Gm_TO | 61 | 39 |
Control (disinfected and wounded) | 0 | 100 |
Control (not disinfected, wounded) | 0 | 87* |