Phytopathology

1. Molecular characterization of phytopathogenic bacteria and fungi
2. Detection of phytopathogenic bacteria by PCR
3. Distribution of effector genes in populations of phytopathogenic bacteria
4. Use of antagonistic bacteria to control phytopathogenic microrganism
   
5. Susceptibility of tomato cultivars to phytopathogenic bacteria and viruses
   
6. Use of natural compounds to control phytopathogenic fungi
   

Molecular characterization of phytopathogenic bacteria and fungi

• Erwinia carotovora (Pectobacterium carotovorum) is the causal agent of soft rot on a wide range of plants. It is divided in five subspecies.
  Twenty-seven isolates of Erwinia carotovora subsp., isolated from soft rot of artichoke plants collected at Piana del Sele (Salerno Province), were tested for pectinolytic activity on crystal violet pectate medium (CVP) and by in vitro inoculation on pepper fruits. Moreover, all the isolates were characterized for the following biochemical and molecular assays: utilisation of α-methylglucoside, production of reducing substances from sucrose, lactose acidification, growth at 36°C and amplification, by polymerase chain reaction (PCR), of a 434 bp fragment of a pectate liase (pel) encoding gene. On the basis of biochemical tests the majority of the collected strains belong to the carotovora subspecies. The isolates gave the expected amplicon of 434 bp by PCR assay. The molecular characteristics of the 27 isolates were investigated by repetitive polymerase chain reaction (rep-PCR) , using 'REP' and 'ERIC' primers. A total of 15 and 14 aplotypes were obtained respectively. Results showed up to eight different aplotypes from samples collected in the same field and three eterologous aplotype from the same plant. Conversely, the same aplotype was recovered from three different fields. Molecular characterization by M13-PCR and AFLP is in progress.
  
  
• Xanthomonas campestris pv. campestris (X.c.c.), is the causal agent of black rot (or vascular blackening) of crucifers.
  To evaluate genetic variability of X.c.c., preliminarily were characterized, for DNA polymorphism, 29 out 150 isolates obtained from different crucifers plants collected from different localities at central and southern Italy. The characterization, performed by PCR with ERIC and BOX primers, distinguished the isolates in 8 and 11 aplotypes respectively. Using M13 primer, the number of aplotypes was almost equal to the number of the isolates. These results indicate a large genetic variability of the population of X.c.c.
  
  
• Fusarium solani (complex) and Fusarium solani var. coeruleum are important causal agents of crop diseases as dry rot of potato tubers during storage. The distinction between F. solani and F. solani var. coeruleum is based on the cultural trait of the pigmentation, unstable character. The cultural, morphological and molecular characterization of 9 isolates of F. solani var. coeruleum and 25 isolates of F. solani obtained from potato, various crops and from soil, shown that unequivocal distinction between the two taxa is possible by molecular methods as AFLP or PCR on minisatellite and microsatellite regions. Moreover, a large variability within the population of these fungi was observed: infact, F. solani is a complex of at least fifty species lineages as reported by DNA sequences of ITS region by other researchers. For these reasons, the taxonomic distinction between F. solani var. coeruleum and F. solani is debatable.
  
  
• Taxonomy of terverticillate Penicillium spp. (subgenus Penicillium) ascribed to the aurantiogriseum group is quite controversial. Many species have been better characterized, or reported in synonymy, or separated from other taxa on account of both morphological and biochemical features. These criteria have often proved to be unsatisfactory, because different laboratories provide different reports on classification of isolates in this group. Moreover, the absence of perfect stages complicates the task. Omology sequence analyses performed on the 5.8 r-DNA-ITS regions and on 5' end of β-tubulin gene sequences, showed 1÷2% and 4÷10% nucleotide variability for 5.8 r-DNA plus ITS regions and for β-tubulin gene respectively. RFLPs performed by software-analyses on these sequences have allowed the selection of some restriction enzymes potentially useful as diagnostic methods. We have evaluated genetic relatedness within a sample of 41 isolates belonging to different species of the aurantiogriseum group.
  PCR-RFLP performed with Sma I on ribosomal regions, permitted only to distinguish P. chrysogenum in respect to all the isolates of the aurantiogriseum group. Using M13-PCR, P. chrysogenum fell in an unique cluster whereas the isolates of the aurantiogriseum group fell in different clusters, confirming in part the distinction in different Penicillium spp. for this group. PCR-RFLP on β-tubulin gene is in progress.
  

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it

Detection of phytopathogenic bacteria by PCR

• A rapid molecular detection method based on the PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primers were designed on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island. A 532 bp amplification product corresponding to an internal fragment of hrpZ_Pst was obtained from fifty isolates of P. syringae pv. tomato, three of them coronatine-deficient strains. No amplicons were obtained from about one hundred among pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and from seventy five unidentified bacterial saprophytes isolated from tomato plants. The method was also tested using leaf and fruit spots from naturally infected tomato plants and asymptomatic nursery plants and seeds of tomato artificially contaminated. The results confirmed high specificity of this detection methods.
  
  
• A rapid molecular detection method based on the PCR amplification of Xanthomonas campestris pv. campestris (X.c.c.), causal agent of the vascular blackening of crucifers, was developed. Two pathovar-specific oligonucleotide primers, designed on the hrcC gene of the Hrp_xcc pathogenicity island, were tested and a PCR protocol was applied on DNA from several bacterial isolates. The expected amplification product of 519 bp was obtained from all the 25 isolates of Xcc. tested. No amplicons were obtained from about 30 isolate of various phytopathogenic bacteria (Xanthomonas spp., Pseudomonas spp., Erwinia spp.) and from epiphytic bacteria of crucifers. Amplicons were obtained directly from naturally symptomatic leves.
  
  
• A molecular detection method by PCR-RFLP of Xanthomonas vesicatoria is in progress.

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it

Distribution of effector genes in populations of phytopathogenic bacteria

• Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck of tomato. Resistance to Pst is conferred by Pto gene, that interacts in a gene-for-gene manner with Pst strains harbouring the avrPto gene. In the year 2002, heavy attack of Pst was observed in the field at ISCI of Battipaglia on some resistant cultivars of tomato. Loss of resistance is probably consequence of loss or mutations of the correspondent avrPto gene with consequent no-recognition by the Pto gene and disease response. Various avirulence proteins can function as virulence factors when the host is not capable to detect them. Because isolates of phytopathogenic bacteria generally contain only one or few avr genes, it can be assumed that virulence is provided by sets of genetically redundant effector genes. Effector proteins, as avirulence proteins, are secreted into the host cell by Type III secretion system. P. syringae secretes/translocates effectors of largely unknown function to largely unknown sites of action to subdue plant cells. Four Pst strains, three isolated from resistant and one from susceptible cultivars in the field at Battipaglia, were analysed for 41 effector genes of the strain DC 3000 of Pst. The four isolates had higher virulence in respect to DC 3000 and did not have 13 effector genes.
  
  
• In collaboration with the Department of Molecular Genetics and Cell Biology of the University of Chicago (USA), analyses of effector genes were made and are in progress on isolates of Pseudomonas syringae pv. tomato (P.s.t.) and other Pseudomonas syringae pathovars collected in different world areas. In particular, it was observed that some isolates of national and international collections isolated from tomato and classified as P.s.t. were very similar to strains of Pseudomonas syringae. The virulence of P.s.t. isolates that show different effector genes composition are in progress on different tomato cultivars.
  

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it

Use of antagonistic bacteria to control phytopathogenic microrganism

• The characterization of antagonistic bacteria, principally isolated from potato tubers and tomato rhizosphere, was performed and is in progress at ISCI of Battipaglia,. Over 200 isolates of bacteria, collected from cultivated fields in Campania Region, were evaluated for their ability to inhibit, in vitro, the growth of Rhizoctonia solani and Fusarium solani. Thirty-one bacterial isolates were chosen for their antagonistic activity and analysed for DNA polymorphism. Ten most active and genetically different isolates, were chosen for in vivo test on potato shoots. The isolate 3 R II bac gave the best control against Rhizoctonia solani ( 63,9 % disease control) and increased plant height and number of tubers compared with untreated control and Rhizoctonia inoculated plants. The isolate 7 IV bac gave the best control against Fusarium sambucinum (51 % disease control) and increased plant height compared with Fusarium inoculate plants. The two isolates able to control these two diseases were gram positive and produced endospores (Bacillus spp.); moreover the isolate 3 R II bac produced volatile substances that inhibits the growth of Rhizoctonia solani. Experimental field trials of biological control on potato are in progress using these selected Bacillus spp. isolates.
  
  
• A lot of studies inferred the use of antagonistic fungi and/or bacteria in the soil, to control soil-borne plant pathogen. Conversely, a minor number of studies inferred the use of antagonists directly on the air organs of the plants. In a research performed at ISCI of Battipaglia, a preliminary characterization of antagonistic bacteria, isolated from tomato phylloplano, was performed. From ten leaf samples, collected from different genotypes of tomato cultivated in two localities of Campania Region, 53 bacterial isolates were chosen. Twenty-one, the greater number genetically different as resulted by DNA polymorphism analyses, were able to inhibit, in vitro, the growth of Alternaria spp. In particular, five of them, were able to reduce considerably (from 58 % to 35 %) the fungal growth. The 21 isolates were all, except one, gram positive and, the greater number, able to produce endospores (Bacillus spp.). At least three of them were able to produce winged substances that inhibits the growth of the fungus and, for other three isolates, the production of siderophores was observed. The bacterial isolates most active in vitro and genetically different, will be evaluated in greenhouse and in the field.

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it

Susceptibility of tomato cultivars to phytopathogenic bacteria and viruses

• Pseudomonas syringae pv. tomato (P.s.t.) cause bacterial speck of tomato. The damage concern quality of the fruits (necrotic spots on the skin) used as fresh or transformed as peeled tomato. Tomato cultivars resistant to P.s.t. are available. The susceptibility of 12 different tomato genotypes to natural infections of P.s.t., was evaluated in the field. Phytopathometric analysis, showed on leaves collected 4 weeks after transplanting slight or not attack for 4 genotypes (S. Marzano 2, Ranco F1, Hypeel 244, Piccadilly F1) whereas for other 4 genotypes (Corbarino ISCI 05, Vesuviano, Faino F1 and Principe Borghese), the attack was intense (from 10 % to over 14 % of diseased leaf surface). At harvesting, the higher incidence and intensity of attack was observed on the fruits of the genotypes Vesuviano and Faino F1, the most attacked before bloom and the most early. The genotypes that not showed damages on fruits were S. Marzano 2, Sorrento, Cuore di Bue and Ibrido Insalataro, slightly or meanly attacked before bloom and with mean-late or late cycle. The resistance of the early genotype Piccadilly F1 was very high.
  
  
• Viruses are the biotic factor mainly limiting the cultivation of tomato. Cucumber Mosaic Virus (CMV) is the most diffused virus in Italy, anywhere tomato is cultivated. In the last years, especially in southern Italy, Tomato Spotted Wilt Virus (TSWV) cause damages too. The control against these viruses is possible using resistant or tolerant genotypes. In the field conditions and during two years (2000 e 2001), the incidence of viral damages on 36 different genotypes of small tomato cultivated in Campania Region was evaluated. Eighteen genotypes with incidence of damaged fruits lower 5 % in 2000, showed incidence of damage not highest 10 % in 2001. For six genotypes (5 S.C-SA., Principe Borghese, Small Fry V.F.N., Grappolino, Lilliput V.F.N. F1, Remo), the lowest incidence (< 5%) registered in 2000, was confirmed in 2001.

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it

Use of natural compounds to control phytopathogenic fungi

• Plants elaborate a lot of compounds useful for pharmaceutical and phytotherapeutic uses. Saponins are glycosides occurring in more than one hundred plant families; the species belong to Asteraceae family, produce high level of triterpenoid saponins which are isoprenoidal natural products with notable biological activities (antitumor, antifungal, antibacterial and antiviral effects).
  Biological test performed with crude leaf extracts of Aster sedifolius against Trichoderma viride and Botrytis cinerea, showed growth inhibition of fungal mycelium. Three purified triterpenoid saponins with oleane-type skeleton, isolated from leaves of Aster sedifolius, were evaluated for their propriety to inhibit growth, in vitro, of phytopathogenic fungi and bacteria. The growth of Rhizoctonia solani, Sclerotinia sp., Fusarium solani and, especially, Sclerotium rolfsii, was significantly reduced; the growth of Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. vesicatoria was inhibited too. One of these saponins is particularly active to inhibit fungal growth.
  
  
• Purification of bacterial filtrates able to inhibit the growth, in vitro, of phytopatogenic fungi, are in progress to obtain the active chemical compounds.
  

Contacts:
Massimo Zaccardelli, m.zaccardelli@isci.it