Background

Nematode is a kind of lower invertebrates distributed widely which can survive anywhere on the earth. According to Hyman (1950) and Poinar (1983)’s estimation, there are over half a million kind of nematodes all over the world. Most soil nematodes are beneficial insects whereas some of them are important plant causative agents so-called plant-parasitic nematodes. Plant-parasitic nematodes have been up to over 5000 species of 200 genus^[1]. Among them, over 100 species of nematodes are damaging the agriculture, forestal and economic crops of China who widely parasitize the roots, stems, leaves, flowers, buds and seeds of manifold plants, harming the development of agriculture and forestry seriously.

According to incomplete statistics, the losses caused by pathogenic nematodes can reach \($\)100 billion every year worldwide^[2]. Meanwhile a newest research by USA indicates that the plant-parasitic nematodes’ damage leads to about \($\)8 billion dollars’ loss to croppers, which takes 12% of the whole value of the crop output. Chinese researchers have done some relevant surveys as well. According to incomplete statistics, 17 provinces such as Anhui, Hainan, Hubei, Gansu, Zhejiang and Fujian have reported root knot nematode (Meloidogyne spp.) disease once, among which the morbidity of some severe regions in Shandong province can up to 2/3^[3]. Pine wood nematode (Bursaphelenchus xylophilus) dose severe harm to forestry and have caused billions yuan losses directly and indirectly. As Fig.1 shown below, 3rd announcement of China’s State Forestry Administration (SAF) in 2015 shows the pine wood nematode disease distribution in China in 2014. Therefore we can note that plant-parasitic nematodes have brought out severe loss to global agriculture and forestry already.

Plant-parasitic nematodes are usually too tiny to catch sight of with naked eyes. In addition, the present surveys about the agricultural damage of nematodes are so limited that people often fail to notice and prevent nematodes in time. The plant-parasitic nematodes are furnished with scalpellus functioning to stab into the plant cells to obtain nutrients after seeking out the host with the amphids on their forehead. As a result, nematodes could damage the host as well as bring in pathogenic fungus and therefore causing complex harm to them. Up to now, the pathogenesis of plant-parasitic nematodes can be concluded in theory as follow:

1. The nematodes give raise to severe mechanical injury to the host plants when feeding on them with the scalpellus.
2. Other pathogens accompanied with the nematodes induce plant disease.
3. Some nematode secretion is toxic to the plants which as a consequence damages them.

In most cases, the mechanical damage by nematodes to host is a drop in the bucket, therefore the latter two theories are relatively common^[4].

The majority of harmful nematodes in agriculture and forestry belong to Tylenchida while a little belongs to Dorylaimida. Meloisogyne spp., Heterodera spp., Aphelenchoides composticola, Ditylenchus dipsaci are nematodes that gravely damage plants around the world. Among them, Meloisogyne spp. mainly destroy the roots of plants by forming root-knot to hinder their development, which in turns, causes the roots rot.(Fig. 2a) According to statistics, a slight Meloisogyne spp. disease can lead to a 20%~30% output reduction, and 50%~70% even a total loss if it’s severe. Heterodera spp. also target the roots of plants primarily, weakening and decomposing the roots.(Fig. 2b) Ditylenchus dipsaci damage the underground part such as the tuber, tuberous root, bulb of the plants which results in their rot and malformation while some local part over ground would also be influenced to turn to malformation,(Fig. 2c) Aphelenchoides composticola such as Bursaphelenchus xylophilus and Aphelenchoides besseyi generally aim at the overground part of plants who infect the leaves and causing lesion and leaf tip drying, and more than that, infect the trunks to kill the whole plant rapidly.(Fig. 2d)

In a word, plant-parasitic nematodes cause serious losses to agricultural crops worldwide. The traditional methods based on the use of nematocides and antihelminthic drugs are associated with major environmental and health concerns, so the development of biocontrol agents to control nematodes is of major importance ^[5]. In this case, our project is designed to control the losses caused by nematodes effectively with biocontrol agents; and after the bait-kill system successfully established, we are looking forward to further expansion to other agricultural pests for the sake of achieving our ultimate goal – establishing a database containing attractant base and toxic protein base in allusion to all sorts of agricultural pests.

Our BioDesign

In our project, we designed three modules separately to attract and poison nematodes and then control the biocontrol agents. Module 1: We made E. coli synthesize limonene to lure plant-parasitic nematodes. Module 2: E. coli would kill them with two kinds of toxic proteins Bace 16 and MpL. Module 3: we introduced a photo-regulated bi-direction transcription system to the whole system, by which means we would be able to regulate the expression of attractant and toxic proteins through the control of light. That’s to say, the E. coli would express attractant to lure nematodes when exposed to light whereas express toxic proteins to kill them as a result of the promoter’s reverse in the dark.

Module 1：Bait MORE ›

Limonene（Fig. 3） is a monoterpenoid with two conformations in the nature, d-configuration and l-configuration. Limonene can attract root knot nematodes of citrus (Tylenchulus semipenetrans) or other plant parasitic nematodes. Our design based on the following two limonene synthase sequences which are named Citrus unshiu CitMTSE1 (GenBank number: AB110636.1) for d-limonene synthase^[6] and Mentha spicata 4S-limonene synthase (GenBank number: L13459) for l-limonene^[7]. At the same time, we also designed another synthase sequence which can synthesize the isoprene GPP, the precusor substance of synthesizing the limonene. And the sequence (GenBank number: AF513112) is from Abies grandis^[8]. We transformed these parts into E. coli to realize the expression of limonene in prokaryotic cell.

Module 2：Killer MORE ›

Bace16 and MpL(Fig. 4) both have a good toxicity toward the nematodes. Bace16 is a kind of serine protease from nematode-parasitic bacteria named Bacillus nematocida. After the expression of Bace16 has completed, the protein can be secreted out of the cell. This proteases could degrade the intestine protein of the nematodes thereby the nematodes will be killed^[9]. MpL is a novel lectin isolated from a kind of parasol mushroom (Macrolepiota procera). It is a kind of intracellular expression protein. MpL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults^[10].