GENERAL ASPECTS TRANSMISSION OF BACTERIA BY SEEDS

Bacteria are widely distributed in nature; they are Prokaryotic microorganisms, they belong to the bacteria domain, proposed based on the comparative sequences of RNA16S or 18S:

All phytopathogenic bacteria are aerobic, and some are facultative anaerobic. They reproduce asexually by bipartition. Their growth depends on nutrition, temperature, humidity, and pH factors. They cause several diseases in plants, with symptoms such as: wilt, rots, and on different plant organs; likewise, they associate with the plant structures of sexual and asexual reproduction.

Bacteria are considered transmitted by seeds when they are carried on or inside them, they penetrate their tissues and remain resting. When the seed is sown, the infection in the new plant will come from the infected seed. Such is the case of the common bean blight (Xanthomonas campestris pv. phaseoli) and of the angular leaf spot of cucurbits (Pseudomonas syringae pv. lachrymans). Other bacteria infect seeds at a superficial level or are mixed with them, such as: Clavibacter michiganensis subsp. michiganensis in chilies, Pseudomonas syringae pv. lachrymans in melons, and Xanthomonas campestris pv. campestris in cabbage, without this meaning that the seeds transmit the disease to the next generation of plants, although it could favor its development in the planting areas.

Bacteria penetrate seeds through the ovary wall, the pericarp, and the teguments, as in Xanthomonas campestris pv. phaseoli in beans; through the testa, as in Pseudomonas syringae pv.pisi in peas; through vascular bundles, as in Xanthomonas campestris pv. phaseoli in beans; or through the micropile as in Pseudomonas syringae pv. lachrymans in melons. Bacteria can also be found in the embryo, as in Clavibacter michiganensis subsp. michiganensis in tomatoes; in the endosperm, as in Pseudomonas syringae pv. lachrymans in melons; in the testa, in the pericarp or in the raphe, as in Pseudomonas syringae pv. pisi in peas.

Bacterial infection can take from an intra-embryo level to a systemic or local level. Or it could take place from the extra-embryo level to local lesions such as Xanthomonas campestris pv. Phaseoli in beans. In squash, Xanthomonas campestris pv. campestris reaches the seeds via the peduncle, silique and funiculus. In peas, the invasion of Pseudomonas syringae pv. pisi ocurrs via sepals and via the peduncle; it migrates to the seed from the pod via the funiculus and to the micropyle in the seed coat. There is a systemic invasion in the tomato or potato seeds by Clavibacter michiganensis subsp. sepedonicus, Xanthomonas campestris pv. vesicatoria in chilies, and Pseudomonas syringae pv. Lachrymans in cucumber. On the other hand, bacteria cause spots in pea seeds by Pseudomonas syringae pv. pisi; and alter the viability and vigor of bean seeds by Xanthomonas campestris pv. phaseoli.

There are epidemiological implications of bacteria related to seeds, which can favor the development of diseases when planting infected seeds, due to the following characteristics: a) prolonged transmission, b) protected form of survival and potential primary inoculant, c) long-distance dissemination and spreading at random, d) preferential selection to strains or races of pathogens, and e) possibility of synergic infections. Many times, bacteria related to seeds cannot be seen by the naked eye, and therefore, moving seed from a region to another without the material undergoing quarantine and without the adequate sanitary tests promotes the generation of new diseases.

These diseases may be explosive and sever if hosts are susceptible and if environmental conditions are favorable. For example, estimates in California concluded that with a population of 1 X 10⁷ ufc of Xanthomonas hortorum pv. carotae per g of carrot seeds, it is enough to start a epiphyte, when environmental conditions are adequate, such as rainfall and temperatures between 25 and 30 °C. When the presence of a disease is found in a critical time period, adequate control measures must be taken. For the control of bacteria transmitted by seeds, fields must be inspected in order to determine the health of the plots, especially when there are conditions that promote the development of diseases that can drastically affect seed production.

BACTERIA ASSOCIATED TO CEREAL SEEDS

Cereals (from Latin Ceres, name of the goddess of agriculture), are very important crops for humanity; approximately 75 % of the total of the surface planted in the world is used for their production. Cereal seeds are a direct source of two thirds of the energy and protein required by humans. Its nutritional value is expressed by its high content of carbohydrates, mostly in the form of starch, as well as proteins, lipids, mineral salts and fibers.

Many different species and varieties of cereals are grown throughout Mexico, maize (Zea mays L. ) being the planted cereal that covers the largest surface in the country, followed by wheat (Triticum aestivum L.), sorghum (Sorghum vulgare L.), barley (Hordeum vulgare L.), rice (Oryza sativa L.), and oat (Avena sativa L. ).

Several bacterial diseases have the potential of being transmitted through seeds, related to cereals of economic importance in certain regions and latitudes of the world, which are not regulated to avoid their introduction into Mexico, or are in this regulation process. Within this context, infections of bacterial etiology have been cited, such as Pseudomonas syringae pv. coronafaciens in oat and sorghum; Xanthomonas translucens pv. undolosa in wheat and barley; Xanthomonas translucens pv. hordei in barley; Xanthomonas campestris, Pseudomonas syringae pv. syringae and Dickeya zeae in maize; Acidovorax avenae subsp. avenae in oat, maize, barley, sorghum, wheat, and rice; Erwinia rhapontici in wheat; Burkholderia andropogonis and Xanthomonas campestris pv. holcicola in sorghum, maize, and others.

In Mexico, in a recent study found that Burkholderia gladioli, Pantoea ananatis, and Acidovorax avenae subsp. avenae causes foliar spots in a maize cultivar in the state of Veracruz. However, the potential for transmission by seeds of these bacteria was not established, although there was information on the sporadic appearance of these symptoms in different growing cycles. It is known that Burkholderia gladioli is related to rice and oat seeds, that Pantoea ananatis is related to onion seeds, and Acidovorax avenae subsp. avenae is to maize seeds.

The economic importance of these diseases varies between regions, countries, and continents. The transmission and spreading of bacteria via seeds, and the high risk it represents in the re-emergence of bacterial seeds or in their introduction to new crop-producing areas, highlights the importance of having faster and more efficient detection schemes and protocols. Determining whether a seed lot is completely free of bacteria that infest or infect them is considered very difficult and improbable. However, it is possible to certify that a seed lot contains a threshold below a specific level of infection by means of seed analysis and the inspection of fields in which they are produced. This has led, in recent years, to emphasizing the development of new detection methods with the aim of increasing the sensitivity and selectivity of different dependant and independent planting techniques, to determine the safety of seeds.

It is considered necessary that in the future the knowledge of the seed-bacteria interaction must be deepen and increased, mainly in the biology of seed infections by bacteria, in the process of colonization, and its epidemiology, as well as in the development of new tests in seed, combining different technologies, understanding the infection thresholds in relation to the sample size and the efficiency of these plant health tests.

BACTERIA RELATED TO VEGETABLE SEEDS

In recent years, vegetable cultivation has become considerably important worldwide. During the period from 2000 to 2006, the Mexican countryside produced an average of 9.33 million tons; from 2006 to 2010, the volume grew 8.6%, which contemplates the intensive use of inputs, particularly for the production of seeds.

Mexico's privileged geographic position gives the country a diversity of climates and ecosystems that not only favor it for the production of vegetables all year long, but also to produce approximately 70 varieties. The different vegetables produced in Mexico are classified into 7 groups, some of the most important of which are: seeds-grains (peas and green beans), fruits (tomatoes, chilies, eggplants, watermelon, melons, and y chayote), bulbs (garlic, onions, and leeks), leaves (Brussels sprouts, broccoli, spinach, chard, and lettuce), tender stems (chicory, endives, celery, and asparagus), pepos (zucchini, pumpkin, cucumber, and black-seed squash), roots (carrots, radishes, beets, beetroot, potatoes, and turnips), and edible flowers (artichokes, pumpkin flower, broccoli, and cauliflower).

One of the characteristics this group of vegetables has in common is that they propagate by botanical seeds, which play a relevant part in the health of the crops. The main form of spreading and survival of most bacteria that affect vegetables is via the seeds, therefore, before its purchase and establishment, one must be sure that it has undergone the highest sanitary controls.

When environmental conditions are not favorable for the growth and reproduction of bacteria, not only does the seed provide protection, but also helps break barriers, both natural and legal, and reach places very far from their place of origin.

It is important to point out that the percentage of transmission by seeds is low, generally fluctuating between 0.1 and 5 %. However, when environmental conditions are adequate for the bacteria, one infected seed in a lot of 10,000 is enough to cause losses of 100 % if the adequate sanitary measures are not taken. In crops in which there is transplantations and require agricultural practices such as defoliated, removing suckers, trimming, and others, the spreading of pathogens will be favored if a control is not carried out. When infected seeds are established, damages can occur in plantlets or in adult plants, and can manifest themselves as rotting, leaf spots, cankers, scabs, wilting, and overgrowth.

Bacteria that affect vegetables belong to the genuses Pectobacterium, Dickeya, Pseudomonas, Xanthomonas, Burkholderia, Acidovorax, Curtobacterium, and Ralstonia. Among these, we can mention:

Seeds-grains (Pseudomonas syringae pv. pisi and Xanthomonas campestris pv. phaseoli).

Fruits (Xanthomonas gardneri, Acidovorax avenae subsp. citrulli and Ralstonia solanacearum).

Bulbs (Pectobacterium carotovorum, Burkholderia cepacia and Pantoea ananatis).

Leaves (Xanthomonas campestris pv. Campestris and Pseudomonas cichorii).

Tender stems (Pseudomonas syringae pv. apii).

Pepos (Pseudomonas syringae pv. lachrymans and Xanthomonas campestris pv.cucurbitae).

Roots (Xanthomonas hortorum pv. carotae, Pectobacterium carotovorum, and Xanthomonas beticola).

Edible flowers (Dickeya chrysanthemi).

METHODS FOR FINDING BACTERIA IN SEEDS

Due to the international economic importance of the activity of producing seeds, the bacterial diagnose has enormous relevance for the timely production of different species in favorable economic conditions. The analysis of seeds helps to detect adequately the bacteria related to them; treatments help protect them from these pathogens and, in some cases, to eradicate them.

Potentially seeds can transmit any bacterial disease; they generally infest the surface of seeds, yet those that cause vascular or systemic infections frequently infect the covers or internal tissues of the seed. The bacteria that infest seeds can remain alive for a limited amount of time, whereas bacteria present inside seeds are extremely long-lived.

Detecting bacteria is a very important filter, which makes it necessary for the diagnose to include several detection methods with sensitivity and efficiency to determine the presence or absence of bacteria. The detection and diagnose of bacteria in seeds is a difficult process, since there is often small amounts of seed for analysis. Also, the inoculant is found infesting or infecting the seeds in low amounts; percentages of contaminated seeds are generally 0.1 % or less. Unfortunately, data for many bacteria on the natural inoculant densities in seeds and the numbers of infected seeds necessary for the development of the disease in the field, are not available.

Some of the methods used include traditional techniques such as the isloation of baterias in in vitro cultures, and their physiological and biochemical characterization. Obtaining bacteria from seeds depends on their percentage of recovery, their release, and on the reduction to a minimum of saprophytic organisms. The isolation methods must focus on achieving the growth of the bacteria, even with a reduced number of bacterial cells. To obtain them, different techniques are used, including sterile distilled water, salt solutions at different concentrations, buffer solutions enriched with culture medium and detergents. Once the strains are isolated, the physiological and biochemical characterizations can be carried out.

Very sensitive methods have currently been carried out that greatly help detect bacteria, even under low concentrations, such as serological methods, particularly ELISA and molecular techniques such as PCR, with the sequencing of the resulting product.

The ELISA (or Enzyme Linked Immuno Sorbent Assay) technique, is based on the direct or indirect detection of an antigen on a solid phase, using proteins known as antibodies, which, in the presence of a specific substrate, produce a colored reaction, which can be measured using a spectrophotometer.

This technique is reliable and quick in detecting bacteria, due to its high degree of sensitivity, since its detection limit is 10 bacteria for every 10 g of sample. However, the technique must be carried out carefully, since reactions may take place with plant residues, saprophytic bacteria, and residues of agrochemicals for treating seeds, which could give false positive results.

PCR (Polymerase Chain Reaction) is one of the most widely used techniques nowadays, with an analysis from a DNA sample, which contains the sequence to be amplified. The amount of DNA needed to carry out this amplification is very small, hence its main advantage. In the detection of bacterial PCR in seeds, the DNA sample must be purified or the DNA extraction must be carried out from the seed sprout or the isolated bacteria in a pure culture. This is in order to avoid that the reaction can be inhibited by compounds such as starch from the seed.

REGULATION IN THE MOVEMENT OF SEED EXPORTS

The applicable plant health legislation is especially transcendent in the normative food and agriculture framework, and it becomes a basic condition for the commercialization of Mexican agricultural products. The measures to protect plant health include applying the plant health measures of Laws, Regulations, Prescriptions, and Procedures established by a particular country to protect the lives of people, animals, and plants from any pollutant or pathogenic organism.

The International Plant Protection Convention (IPPC) is an international plant health agreement with 173 member countries to date, the goal of which is to protect planted and wild plants, preventing the introduction and spreading of pests. The work program of the IPPC Secretariat focuses on the creation of International Standards for Phytosanitary Measures (ISPM), the exchange of official information, and the creation of capability and technical assistance. These ISPM are created as part of a worldwide policy and technical assistance program in matters of quarantine carried out by the FAO. All IPPC signing countries are obliged to have a national phytosanitary certification system, according to indications specified in the Text of the Convention and in the ISPM No. 7 "System of Phytosanitary Certification." ISPM No. 12 "Phytosanitary Certificate" was also created, which mentions that the purpose of the Phytosanitary Certificate (PC) is to indicate that the shipments comply with the phytosanitary requirements established by importing countries, and must be issued to this end.

The PC is an official document issued by SAGARPA, which confirms the compliance of the legal orders in matters of Plant Health, which the exports of plants, their products and subproducts adhere to when being a possible phytosanitary risk for the importing country. This PC is issued after verifying the place of origin of the product. Official staff or a Verification Unit can carry this out. The verification consists in confirming that the shipment complies with the phytosanitary requirements established by the importing country, which could include the revision of documents, sampling, and lab diagnosis, the results of which must be grounded in a verification verdict, which, along with the corresponding payment of rights, are the basis for the issuance of the PC.

To know the phytosanitary requirements to be complied in order to ship plant materials, their products and subproducts, the legislation of the importing country must be consulted, and verify that it is established in an Work Plan or Agreement, Addendum, Import Manuals or in import permits. In Mexico, bacterial pathogens are regulated to prevent their introduction to the country by Mexican Official Regulations, such as NOM-013-FITO-1995 in rice for pathogens Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. Oryzicola, NOM-017-FITO-1995 in wheat for pathogens Pseudomonas syringae pv. atrofaciens, and NOM-018-FITO-1995 in maize for Clavibacter michiganensis subsp. nebraskensis, Pantoea stewartii, and Pseudomonas andropogonis.